Distal to Proximal—Functional Coupling in RNase P RNA ...444297/FULLTEXT01.pdf · RPP protein subunit of the RNase P ... is the molecule that carries the genetic information in

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List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Brännvall, M., Kikovska, E., Wu, S. & Kirsebom, L.A. (2007). Evi-

dence for induced fit in RNase P RNA mediated cleavage, J. Mol. Bi-ol. 372, 1149-1164.

II Wu, S., Chen, Y., Lindell, M., Guan Zhong, M. & Kirsebom, L.A. (2011). Functional coupling between a distal interaction and the clea-vage site in bacterail Rnae P RNA mediated cleavage. J. Mol. Bi-ol.411, 384-396

III *Sinapah, S., *Wu, S., *Chen, Y., B. M. F. Pettersson, V. Gopalan & Kirsebom, L.A. (2010). Cleavage of model substrates by archaeal RNase P: role of protein cofactors in cleavage-site selection. Nucl. Ac-ids Res. 39, 1105-1116.

IV Wu, S., Kikovska, E., Lindell, M. & Kirsebom, L.A. (2011). Cleavage mediated by the catalytic domain of Rnase P RNA. Manuscript.

V Wu, S., Chen, Y., Guanzhong Mao, Trobro, S., Kwiatkowski, M. & Kirsebom, L.A. (2011). The -1 residue and transition-state stabiliza-tion in RNase P RNA-mediated cleavage. Manuscript

* Shared first authorship Reprints were made with permission from the respective publishers.

Contents

Introduction.....................................................................................................9 Divalent Metal Ions / Mg2+.......................................................................11 Processing and Maturation of tRNA ........................................................14 RNase P ....................................................................................................16

RNase P and the RNA subunit.............................................................16 Different types of RNase P and the conserved parts within them .......17 Substrates of RNase P..........................................................................18 Divalent metal ions and RNase P ........................................................19 Interactions between RNase P RNA and its substrate .........................21

Different model substrates used in my study ...........................................25 A glance at the kinetics ............................................................................26 About this thesis .......................................................................................30

Induced fit model ..........................................................................................31

The two domains of the RNase P RNA ........................................................37 Contribution of the S-domain...................................................................37 Contribution of the C-domain ..................................................................40

What is happening around the cleavage site? ...............................................43 The contribution of the -1 residue in the substrate ...................................44

Concluding remarks ......................................................................................49

Swedish Summary ........................................................................................51

Acknowledgements.......................................................................................54

References.....................................................................................................56

Abbreviations

A adenine C C5 DNA

cytosine Escherichia coli RNase P subunit deoxyribonucleic acid

G guanine RNA ribonucleic acid RNase P ribonuclease P RPR RNase P RNA RPP protein subunit of the RNase P M1RNA Escherichia coli RNase P RNA PFU Pyrococcus furiosus RNase P pre-tRNA precursor tRNA tRNA transfer RNA U uracil

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Introduction

As is well known, DNA (Deoxyribonucleic acid) is the molecule that carries the genetic information in almost all organisms’ cells (with the exception of RNA virus). The inherited genetic information is then transcribed to another molecule in cell, RNA (Ribonucleic acid). By intricate processes, RNA is then translated into a more complex molecule, protein. This process from DNA to RNA to protein is called the central dogma of molecular biology (Figure 1). Figure 1. Central dogma of molecular biology. The genetic information carrier DNA is transcribed to RNA: RNA can also be reverse-transcribed back to DNA. RNA is then translated to protein.

Proteins are very important molecules in organisms. They do the most of the work, for example in the human cell. Proteins can be hormones, enzymes, cell signals and they can establish the structure of our body and more. None-theless all the information that encodes protein is carried by DNA. DNA carries the biological information that is inherited from parental cells and DNA that is not for the genetic coding can have structural purpose or be involved in regulating gene expression. DNA is a more stable molecule compare to RNA because it has –H instead of –OH at position 2’ on the sug-ar ring and it usually has two complementary strands. However, from studies in recent years RNA actually appears to be a very important molecule in its own right and not a disposable copy of the DNA. In fact, RNA is considered a more ancient biological molecule compare to DNA and protein. There is much evidence supporting this hypothesis, which is called “the RNA world” (Gesteland et al. The RNA World, Third edition). No matter what happened in the past, RNA is still serving paramount roles in cellular processing as we discovered in recent years. The discovery of Ri-bozymes (see below), Riboswitches (Bastet et al. 2011) and RNA interfer-

Transcription

Reverse-Transcription

Translation

DNA RNA Protein

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ence (RNAi) (Ketting 2011; Perrimon 2010), are among the important dis-coveries that have opened up the era of the RNA world. Table 1. List of known naturally existing ribozymes.

Name of the ribozyme Found presenting species Biological function Divalent Metal

ion depended

Peptidyl transferase 23S rRNA All species Decoding mRNA into

protein Yes

RNase P Almost all species tRNA processing Yes

Group I introns In bacteria, higher plants, phage and lower eukaryotes

Intron splicing Yes Large

ribozymes

Group II introns In plants, fungi,

bacteria, and lower eukaryotes

Intron splicing Yes

GIR1 branching ribozyme

In myxomycete Didymium iridis and several species of the amoebaflag-ellate Naegleria

Twin-ribozyme intron organization Yes

Leadzyme In 5S rRNA of

cellular mammal-ian ribosomal

Lead toxicity Yes

Hairpin ribozyme In RNA satellites of plant viruses

Replication of the satellite RNA No

Hammerhead ribo-zyme

In plant viroids, helminths, ar-

chaebacteria and eubacteria

Satellite RNA and Viroid RNA excision No

HDV ribozyme In virus Viroid RNA excision Yes Mammalian CPEB3

ribozyme In mammals mRNA processing No

VS ribozyme In mitochondrial

DNA of Neurospo-ra

Satellite RNA excision No

glmS ribozyme In some Gram-positive bacteria

Gene regulation/ riboswitch No

Small ribozymes

CoTC ribozyme Eukaryotic Transcription termina-

tion of RNA poly-merase II

Yes

Ribozyme (ribonucleic acid enzyme) is a key finding that opened the RNA world hypothesis that was first named by Kelly Kruger and colleagues in 1982 (Kruge et al. 1982). Ribozyme is a kind of enzyme. Unlike protein enzyme, the catalytic subunit of ribozyme is made of RNA. Actually the ribosome, where the protein is translated from the mRNA code, has RNA as the core active site (Nissen et al. 2000). Furthermore it has been shown that a

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highly deproteinized ribosome is still active (Noller et al. 1992). Thus the ribosome is indeed a ribozyme. In nature ribozymes can either catalyze the hydrolysis reactions on their own phosphodiester bonds or on phosphodiester bonds of another RNA, with or without help of the protein subunit. Some of the ribozymes can also catalyze the reverse reaction, for example VS ri-bozyme (Lilley, 2011). In vitro selected ribozymes have also been reported to be able to catalyze peptide bond formation (Tamura 2011; Zhang et al. 1997). Already known naturally existing ribozymes are listed in table 1.

Divalent Metal Ions / Mg2+

Metal ions play a very important role in the catalysis of the ribozymes. For a better understanding of ribozyme catalysis, it is essential to talk about metal ions, especially divalent metal ions in big ribozymes. Divalent metal ions are essential for enzyme activity in the cell. In the cell one of the most abundant divalent metal ion is Mg2+. It exists in all cell types and all organisms. The concentration of Mg2+ in animal cells is 30 mmol/L (Ebel et al. 1980). In plant, the concentration is higher. Over 300 enzymes need the presence of Mg2+ for their catalytic activity. These enzymes appear in all metabolic pathways. For example, ATP and T7 RNA polymerase are all Mg2+-dependent (Yang et al. 1980). In general, Mg2+ is involved in cata-lytic activity by interacting with the substrate or the enzyme, or both. Mg2+ interacts with the substrate or the enzyme through inner or outer sphere co-ordination, by altering the conformation of the enzyme, by stabilizing anions or reactive intermediates, by binding to ATP thereby activating the molecule to act as nucleophile and eventually making the chemical reaction occur. Manganese (Mn2+) is a divalent metal ion that can replace Mg2+ in some conditions, for example in enzymes catalysis. Chemically Mn2+ is similar to Mg2+. In our experiment (see below) we also used Mn2+ to replace Mg2+ and it gave interesting results. Divalent metal ions are also important in catalysis of ribozymes. As table 1 shows, most of the small ribozymes do not need divalent metal ions for their catalytic activity. Data suggest that monovalent cations are enough for the catalysis of these small ribozymes. The cleavage mechanism of ribozymes can be called general acid-base catalysis and it involves SN2 reaction. In cleavage mechanism of small ribozymes adjacent internal nucleotides out-side the cleavage site act as proton donors and acceptors. The cleavage prod-ucts become a 2',3'-cyclic phosphate and a 5'-hydroxyl group (Wilson et al. 2011; Murray et al. 1998).

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In contrast, not like some small ribozymes that can only rely on monovalent ions, divalent metal ions are important for the bigger ribozymes, like Group I and Group II introns and RNase P, to have efficient catalysis (Adams et al. 2004; Toor et al. 2008). Therefore these ribozymes are called metalloen-zymes. However monovalent ions are disposable for catalysis of bigger ri-bozymes. For example in RNase P-mediated catalysis, monovalents are needed. Monovalents can also help to shield the negatively charged RNA backbone and facilitate in RNA folding. However, the preference for mono-valent ions and their exact mechanism are still unclear. Divalent metal ions play important roles in ribozyme catalysis in two ways:

1) Folding and stabilizing the structure. Correct folding is crucial for enzymes catalytic activity. The reason why proteins can fold into rigid three-dimensional structure in water-based solu-tion is because they have hydrophobic residues. Nucleic acids do not have the hydrophobic residues to help them fold. Instead nucleic acids have a negatively charged phosphate backbone. This makes the folding of nucleic acid more difficult because of the repulsion effects caused by this backbone. Divalent metal ions can neutralize the negatively charged backbone to some extent and thereby help the folding. Data also show that divalent metal ions can stabilize the structure of the RNA by coordinating to 2’OHs and bases, thus stabilizing the wobbly base pairs and the backbone (Shi et al. 2000).

2) Stabilizing transition state conformations, thereby generating the nu-cleophile and its involvement in the chemistry of the cleavage.

Efficient and accurate catalysis not only depends on the structure of the mo-lecule in a steady state, but also relies on the transition state conformations. Cleavage of RNA is possible because nucleophile attacks the phosphodiester backbone and hydrolyzes the chemical bond. Metal ions are involved in cleavage in various ways: 1). A metal-coordinated hydroxide ion can act as a general base or Lewis acid to abstract the proton or accelerate the deprotona-tion, respectively. 2). Stabilize the 3’-oxygen leaving group. 3). Help the nucleophilic attack by either stimulating the phosphorus center or stabilizing the charged trigonal-bipyramidal intermediate (Yasuomi et al. 2001). Differ-ent large ribozymes have different mechanisms for cleavage, although their chemical mechanisms all involve SN2 displacement reaction. For large ri-bozymes Group I and II introns share a similar general acid-base catalysis mechanism. They both have two-step splicing mechanism. In the first step, in Group I intrions, the 3’-OH group of the attacking external G residue is the nucleophile and attacks on the 5’ splice site. In Group II introns, the 5’

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splice site is attacked by the 2’-OH of the internal A residue or by a hydrox-ide ion. The second step involves an attack from the 3’-OH of the 3’-end upstream exon on the 3’ splice site to make the final splicing products. Metal ions were suggested to serve roles in the reaction by their association with a water molecule. Mg2+ in particular was suggested to coordinate to substrate oxygens, then activate the nucleophile, and stabilize the scissile phosphate and the charge on the leaving group. The two-metal-ion mechanism that was seen in protein enzymes was suggested to be the mechanism of the Group I and Group II intron splicing (Yasuomi et al. 2001; Piccirilli et al. 1992; Stah-ley et al. 2006; Forconi et al. 2009; Steitz and Steitz 1993). In this model, two divalent metal ions that are 3.9Å apart were suggested to facilitate the catalysis. Both divalent metal ions act as Lewis acids that can stabilize the chemical transition state. One ion activates the attacking water or sugar hy-droxyl. Another one is suggested to stabilize the oxyanion leaving group (Steitz and Steitz 1993). RNase P has also been suggested to use a two-metal-ion mechanism in the cleavage reaction. The RNase P-substrate complex structure that was solved recently also suggests that there are at least two distinct metal ions that play an important role in the cleavage (Reiter et al. 2010). However, apart from the roles of the divalent metal ions in the cleavage mechanism, RNase P in general has a different mechanism in catalysis compared to Group I and II intron. In RNase P-induced cleavage, the deprotonized water molecule acts as the nucleophile. It was suggested that the 2’OH at the -1 residue acts as an outer (or inner) sphere ligand for Mg2+ (Persson et al. 2003; Brännvall et al. 2004; Zahler et al. 2005). It is possible that divalent metal ion (Mg2+) coor-dinating 2’OH also uses the 3’ bridging oxygen and the pro-Rp-oxygen as coordinator in RNase P RNA-mediated cleavage. Particularly pro-Rp-oxygen has been observed in type A, type B and eukaryal RNase Ps (Chen 1997; Thomas et al. 2000) (for a description of the different types of RNase P, see below). Divalent metal ions were also suggested to play roles in stabi-lizing and generating the nucleophile, stabilizing the transition states and stabilizing the leaving group (Smith D. et al. 1993; Guerrier-Takada et al. 1986; Warnecke et al. 1996, 2000). The products after cleavage by RNase P remain 5’-phosphates and 3’- hydroxyls. Note that divalent metal ions can also induce hydrolysis of the RNA to yield products with 5’-hydroxyls and 2’;3’-cyclic phosphates. Thus in RNase P-mediated cleavage, RNase P has to prevent the divalent metal ion-induced hydrolysis as a result of a nucleo-philic attack from the 2’-OH side and to favor the cleavage from the other side operated by a hydroxide ion coordinated by a divalent metal ion. How-ever, the exact mechanism of RNase P-mediated cleavage remains unclear (Fig. 2).

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Regarding the divalent metal ions, we were interested in answering the fol-lowing questions: 1) What is the role of the divalent metal ions (Mg2+) in RNase P RNA catalysis? 2) How many functional Mg2+ ions are involved in the catalysis and what are their roles? 3) What is the relevance of Mg2+ in the structure/ formation changes of the RNase P RNA and substrates? In my thesis I will discuss these questions.

Figure 2. A model of reaction catalyzed by RNase P RNA through two-metal-ion mechanism. One divalent metal ion coordinated hydroxyl ion acts as the nuclophile. Another divalent metal ion stabilize the oxyanion leaving group. Two metal ions both act as Lewis acids and stabilize the pentacoordinated transition state. The prod-ucts after cleavage are 3’OH and 5’phosphate.

Because divalent metal ions can induce cleavage on the backbone of RNA and the binding sites of the divalent metal ions are specific, divalent metal ions can be used as tools to map the metal-ion binding site on RNA. Pb2+ has been used frequently in footprint experiments to investigate the structural change of the RNA (Brännvall et al. 2001; Doctor Thesis of Magnus Lindell). In our study we also used Pb2+-induced cleavage in footprint experiments to explore the structural changes of the RNase P RNA or the substrates (see below). For example, we used footprint experiments to explore the confor-mational change of the RNase P RNA after we displaced some nucleotides.

Processing and Maturation of tRNA When the tRNA is transcribed in the cell, it has extra nucleotides on both the 5’ and 3’ ends of the pre-tRNA. These extra nucleotides need to be removed by enzymes so that the pre-tRNA is matured and functional for translation (Figure 4.).

H

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There are several ribonucleases, depending on the origin, involved in the processing at the 3’ end of a pre- tRNA (Schurer et al. 2001 and Mörl & Marchfelder, 2001). tRNase Z is the main enzyme for maturation of the 3’-end (Hartmann, et al. 2009). In bacteria, the endoribonucleases RNase E, RNase G, and numbers of other exonucleases are responsible for tRNA 3’-end processing (Li et al. 1996; 2002). Moreover, for these tRNAs without the encoded 3’-CCA motif or where the 3’-CCA motif is damaged after 3’ maturation, tRNA nucleotidyl transferase adds the 3’-CCA motif to the tRNA (Wen et al. 2005). However, at the 5’-end, RNase P is the only enzyme that is responsible for the maturation of a pre-tRNA. RNase P can cleave on the substrates irrespec-tive of the existence of the 3’end (Tous et al. 2001). RNase P in most of these cases cleaves the substrate between the -1 and +1 residues, except for tRNAHis and tRNASeCys ; these are cleaved between -2 and -1 residues (Orel-lana et al. 1986; Burkard et al. 1988a, 1988b; Green. et al. 1988; Kirsebom et al. 1992). After cleavage by RNase P, the cleavage products become a 3’ –OH and a 5’-phosphate, respectively (as mentioned before). This applies to both presence and absence protein part of the RNase P.

objectives of my thesis work have been to investigate substrate interaction and mechanism of cleavage by the trans acting ri-

bozyme RNase P RNA. First, I will give a general overview of the field and relevant factors that influence cleavage efficiency and choice of cleavage site. This will be followed by a brief summary of the strategy and methods that I have used in my studies. Finally, I will discuss the specific projects during my thesis work.

The

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RNase P RNase P and the RNA subunit There are about 1000 copies of RNase P RNA (also is called P RNA or RPR) in the fast growing E. coli cell and are responsible for tRNA precursor processing in all three kingdoms of life (Dong et al. 1996). In eukaryotes, RNase P is located in the nucleus. The kinetics of RNase P-mediated cataly-sis includes substrate binding, chemical cleavage, and product release, fol-lowed by new rounds of substrate binding. Thus RNase P is a turn-over ri-bozyme. Some of the ribozymes, like the Hammerhead ribozyme, generate cleavage on itself. However, RNase P, like the ribosome and other protein enzymes, has another RNA as its substrate and acts in trans. Therefore RNase P is a true enzyme. Bacterial RNase P has two subunits: one RNA subunit and one protein sub-unit. The number of the protein subunits varies, depending on the origin of the organism. For example, there are four to five protein subunits in the Ar-chaea and at least nine in the Eukarya (Kirsebom. 2007; Kirsebom et al. 2009). It is worth mentioning that in human mitochondria no RNA part of the RNase P has been found so far and protein alone has the RNase P prop-erty (Kufel et al. 2008; Holzmann et al. 2008). This might be very important in terms of evolution. Early, people thought the RNA part of the RNase P was just a scaffold for the enzymatically active protein to attach to and get the right configuration. After an experiment by Cecilia Guerrier-Takada, it was clear that the RNA part of the RNase P had catalytic activity (Guerrier-Takada et al. 1983). In fact, the RNA part of the RNase P is the catalytic subunit of the enzyme. In vitro, most RNase P RNA can perform enzymatic activity without the help of the protein part (Thomas et al., 2000; Kikovska. et al. 2007), but in vivo the protein part is indispensable. It is already known that in bacteria the protein part of RNase P binds on the 5’ leader of the precursor to stabilize the RNase P-substrate complex to in-crease the affinity of the substrate and catalytically important metal-ion binding (Crary et al. 1998; Sun et al. 2006). In bacterial RNase P, the protein part is also involved in product release, prevention of product rebinding, cleavage site recognition, broadening the substrate selection and stabilizing the native structure of RNase P RNA (Tallsjö and Kirsebom 1993; Liu et al. 1994; Park et al. 2000; Buck et al. 2005). However, the recently published co-crystal structure indicates that the protein is near the 5’ end of tRNA, but not in direct contact with it. Instead, the protein sits between P15 and P3 stem of RNase P RNA and also contacts the CR-IV and CR-V regions (Rei-

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ter et al. 2010). There is little known about the function of archaeal and eu-karyal RNase P proteins. Interestingly, the eukaryotic RNase P holoenzyme cleaves on the tRNAHis precursor and leaves 7-bp-long acceptor stem (for a review see Kirsebom and Torobro 2009). This is in contrast to the bacterial RNase P RNA and the human RNase P RNA where the RNase P RNAs gen-erated cleavage leaves an 8-bp-long acceptor stem (Orellana et al. 1986, Kikovska et al. 2007). RNase P is essential for life and exists in all kinds of organisms. It is a uni-versally conserved ribozyme. This implies that RNase P is perhaps a rem-nant of the RNA world and has been important in evolution. However there are still a few exceptional species where no RNase P has been identified. For example, there are no candidates for the genes that code for the RNase P RNA or the protein in Aquifex aeolicus, although evidence shows that some RNA is involved in the RNase P-like performance in this organism (Kaza-kov et al. 1991; Lombo et al. 2008; Marszalkowski et al. 2008). Likewise, in the archaeon Nanoarchaeum equitans, there is no RNase P found (Lennart et al. 2008). There are also some other organisms that do not have RNase P, like viruses or phage, but these organisms cannot grow on their own.

Different types of RNase P and the conserved parts within them

Figure 3. Secondary structure of different types of RNase P RNA from Bacteria and Archaea, except for type P. a) E. coli RNase P RNA. b) Pyrococcus furiosus (Pfu) RNase P RNA. c) Bacillus subtils RNase P RNA. d) Thermomicrobium roseum RNase P RNA. e) Methanocaldococcus jannaschii RNase P RNA. The type of RNase P RNA that each of them belongs to is stated underneath. The picture is based on the figure from RNase P database: http://www.mbio.ncsu.edu/rnasep/home.html

RNase P is an ancient RNA molecule and is considered to be a relic of the RNA world. During evolution Bacteria and Archaea RNase P RNA have

Type A Type B Type C Type M

a b c d e

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evolved into five different types: Type A, Type B, Type C, Type M and Type P. RNase P in Eukaryotes has a more complicated situation and has not been divided to classes. A type means ancestral type. E. coli RNase P RNA, i.e. M1 RNA belongs to this type. Most of the bacteria and archaea RNase P RNAs also belong to this type. B type was named because it was first discovered in Bacillus. It exists in most Firmicutes. C type means the Chloroflexi type. M type means the Methanococci type. This type only belongs to Archaea RNase P RNA. The RNA part of this type RNase P is not proficient in ca-talysis in vitro without the protein part. P type is named from Pyrobaculum. This type has only been seen in the species Pyrobaculum. It has almost no specific domain (S-domain) as in other Bacteria and Archaea RNase P RNAs and it has less conserved parts than other types of RNase P RNA (Lai et al. 2010). (Figure 3) Although the sequences of the RNase P RNA are very different, a core of the sequence and part of the secondary structure are still conserved within all the RNAs. The P8 and L15 are involved in direct substrate binding, and the P4 and surrounding joining regions are the catalytic important regions (Figure 5). These regions are evolutionary conserved in A, B and M types of RNase P RNA, and even in eukaryotic RNase P RNA (Kirsebom 2007). All the RNAs (except for P type) have two domains: specificity domain (S-domain) and catalytic domain (C-domain) (Figure 5). The crystal structures for both type A and type B are available and the structures show that the S-domain of these two types of RNase P RNA can fold into a different tertiary structure (Kirsebom 2007; Haas and Browa, 1998; Kazantsev et al. 2005).

Substrates of RNase P RNase P has many substrates other than precursor-tRNA in nature. These substrates either have a single stranded RNA attached on the 5’end of a long hairpin construct, like 4.5 S RNA (Bothwell et al. 1976), or they have a tRNA-like construct, like tmRNA (Komine et al. 1999) or plant viral RNAs (Guerrier-Takada et al. 1988). Moreover, it is also involved in the maturation of some other noncoding RNAs such like 2S RNA, snoRNAs. In the leader sequence of mRNA it is suggested that RNase P can recognize and cleave riboswitchs such as the coenzyme B12 riboswitch in E. coli and Bacillus sub-tilis (Hori et al. 2000; Tous et al. 2001; Altman et al. 2005; Seif and Altman 2008). Among all these substrates tRNA precursors are the most abundant.

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It was suspected that there had to be more substrates for RNase P in the cell. The way to look for these substrates was to look for the accumulated precur-sors in the cells under the conditions when no RNase P is made (Yang and Altman 2007; Samanta et al. 2006; Coughlin et al. 2008). In this fashion some other potential substrates were found. For example, in some RNAs in E. coli there are cleavage sites between ORF regions for RNase P. These sites were suggested to be important for the degradation or regulation of the operons. Nevertheless, it is not easy to recognize the substrates in nature due to the conformational variation of the RNA in solution (Greenleaf et al. 2008) or due to the transitional conformational changes it undergoes. Many artificial model substrates have been used in RNase P research as well. According to data the minimal size of the substrate in vitro that can be rec-ognized and cleaved by RNase P should contain one extra nucleotide up-stream of the canonical cleavage site, together with a 7-basepair stem and an RCCA motif at the 3’end of the substrate (Werner M et al. 1997; Forster and Altman 1990; Liu and Altman 1996). Although the RCCA motif is not abso-lutely required for cleavage by RNase P RNA in vitro, evidence shows that it is important for the rate of cleavage (Svärd and Kirsebom, 1992). The sub-strate with two nucleotides upstream of the cleavage site has significantly increased affinity to RNase P RNA and a faster cleavage rate compare to the substrate that has only one nucleotide before the canonical cleavage site (Crary et al. 1998). Nevertheless in paper I we report a small RNA, that has only a 3- base pair stem, a 9-nucleotide 5’-leader and a 4- nucleotide loop, which can act as substrate for the RNase P RNA in vitro (Detailed discus-sions below, Figure 7). In addition, there were reports demonstrating that the bacterial RNase P holoenzyme cleaves small, single-strand RNA as well (Loria et al. 2001).

Divalent metal ions and RNase P As mentioned before, divalent metal ions, particularly Mg2+, are essential in RNase P mediated catalysis, both in vivo and in vitro. There are about 100 Mg2+ ions bound to RNase P RNA (Beebe et al. 1996). Some of these Mg2+ ions contribute to RNase P RNA folding or they assist the interaction of the RNase P RNA with its substrate, the interaction of RNA with protein, or they are involved in the chemistry of cleavage. Note that the structural Mg2+

binding site is not necessary important for RNase P catalysis. The functionality of RNA is very much dependent upon its correct folding, it is the case for RNase P RNA and its substrate. Bacterial RNase P RNA is catalytically active without the protein part, so the folding of its RNA part of bacterial RNase P is independent of the protein part of the RNase P as well,

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making the role of Mg2+ even more important. The folding of B. subtilis RNase P RNA in vitro occurs at Mg2+ ion concentrations <3.2mM and the optimal catalytic activity is at >50mM. M1 RNA folds at Mg2+ ion concen-trations <2.4mM and the optimal catalytic activity is at >25mM (Pro-teinRwviews, V.10: Ribonuclease P, Fenyong Liu and Sidney Altman). The role of the divalent metal ion is twofold in RNase P RNA-mediated catalysis. Firstly, the divalent metal ions promote folding of the RNA to an active conformation and secondly, they can promote chemical cleavage. Other than Mg2+, Ca2+, Mn2+, Sr2+ and even Pb2+ can also induce folding and promote cleavage of RNase P RNA to some extent. It is noteworthy that Pb2+ induced folding leads to a different structure than other divalent metal ions. Pb2+ and Sr2+ cannot promote RNase P RNA catalysis at physiological pH by themselves, but together they can (Brännvall et al. 2001). In nature, both Mg2+ and Ca2+ are present in the cell, and possibly they can coordinate to-gether to regulate the RNA-based activity as a function of their intracellular concentration. For instance, Mg2+ can promote a faster catalysis than Ca2+in RNase P-induced cleavage thereby regulating the biological processes in which RNase P is involved. Several important Mg2+ ions have been identified in RNase P RNA. The Mg2+ on the P4 helix does not induce any conformational change of RNase P RNA, but it is believed to contribute to a distal effect in the vicinity of the cleavage site (see above). Divalent metal ions are also important in the TSL-(T-stem-loop region) /TBS (T-stem- loop binding site) interaction (about different interactions, see below). It has been claimed that the interaction of TSL-/TBS changes the conformational position of the divalent metal ion in the vicinity of the cleavage site (Brännvall et al. 2007). The RCCA-/ RNase P RNA interaction also has divalent metal ion involved in. There are three divalent metal ions in the P15-loop (Kazakov and Altman 1991; Kufel and Kirsebom 1996; Zito K et al. 1993; Glenmarec et al. 1996). These metal ions are suggested to stabilize the interaction formation of RCCA-/ RNase P RNA as well as influence the divalent metal in the vicinity of the cleavage site (Brännvall et al. 2004). A divalent metal ion is suggested to stabilize the interaction of +73/294, and play a role in the surrounding of the exocyclic amine of G+1 and 2’OH, N7 of base +73 of the substrate. There are divalent metal ion(s) also in the vicinity of the cleavage site that is suggested to influ-ence the conformation of A248/ N-1 interaction (Brännvall et al. 2007). Additionally, it was suggested that the true substrate of RNase P should con-tain a divalent metal ion in the vicinity of the +1/+72 pair (Perreault and Altman 1993).

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Because of the importance of Mg2+ in RNase P RNA catalysis, determining the Mg2+ profile and the optimal Mg2+ concentration is necessary for finding out the characteristic of each RNase P RNA/substrate combination. Note that cleavage rate increases as the Mg2+ concentration increases before reach a plateau, therefore measuring the rate before the optimal Mg2+ concentration (plateau) makes the data not comparable with each other. In paper II, we used the optimized Mg2+concentration in the cleavage rate determination to make the data more comparable than in paper I (see below).

Interactions between RNase P RNA and its substrate

Figure 4. Secondary and tertiary structure of matured tRNAphe. The secondary struc-ture elucidates the different parts of the tRNA. The tertiary structure elucidates the “L-shape” of the tRNAphe and the corresponding parts of the secondary structure. (The secondary structure is modified from Wikipedia provided picture: http://en.wikipedia.org/wiki/Transfer_RNA )

It has been suggested that the tRNA domain of the tRNA precursor has al-ready folded to its characteristic structure at the precursor stage. RNase P can recognize this structure and interacts with it (Chang 1973; Swerdlow et al. 1984; Kirsebom 2007) There are many interactions between RNase P RNA and the pre-tRNA. These interactions are distributed on the acceptor stem and the T-arm of the pre-tRNA (Kirsebom 2007). The matured tRNA is folded into an L-shape tertiary structure (Kim et al. 1973) and the interactions with RNase P RNA are all located on “upper arm” of the L-shape (see Figure 4).

Cleavege site of RNase P

D/ T loop

RCCA motif

D-stem & anticodon-stem & variable loop

acceptor-stem & T-stem

Anticondon

RCCA motif

RNase P Cleavage site

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Figure 5. The secondary structure of E. coli’s RNase P RNA. The region with the dark circle indicates the conserved regions within different types of RNase P RNA. The dotted line in the middle of the structure is the separation line of the S-domain and the C-domain. The picture is modified from RNase P database, http://www.mbio.ncsu.edu/rnasep/home.html.

These interactions are determinants which RNase P recognizes and binds to result in accurate and efficient cleavage. RNAs that do not have the tRNA like structures e.g., 4.5 S RNA, can still be recognized by RNase P even if they do not have all the determinants (Altman & Kirsebom 1999). The more determinants that the RNase P can interact with, the more accuratly and effi-ciently the RNase P can cleave its substrate (Svärd et al 1993).

S-domain

C-domain

23

The TSL-/TBS interaction T-stem-loop region (TSL-region) is located at the “elbow” of the L-shape tRNA (also referred to as the D/T loop domain). The part of RNase P RNA that interacts with this domain is called TBS (T-stem- loop binding site). This TBS binding site is within the conserved part of the specific domain (P7-P11) of RNase P RNA (Krasilnikov et al. 2004; Pan et al. 1996; and below) (see Figure 5). In B. subtilis, it has been suggested that the interac-tion with TSL is established through tertiary interaction of the 2’-OHs of four residues (residue 54, 56, 61 and 62) and possibly a 4-amino group of residue C57 (Loria et al. 1997) (see Figure 4). In the recently published RNase P-tRNA co-crystal structure, these interactions were confirmed; in particular, residues 56 and 19 in the TSL region interact with TBS region in RNase P RNA (Reiter et al. 2010). In B. subtilis the residue corresponding to A233 in E. coli RNase P RNA interacts with the 2’-OH at residue 64 in the T-stem in the pre-tRNAphe (Pan et al 1995). Residue 118 in the specific do-main is also part of the TBS region (Grandeur et al.1994). Moreover, A129, A130, A180, G229 and G230 (E. coli RNase P RNA numbers) were found to interact with the TSL region from the recently published crystal structure (Reiter et al. 2010). The TSL/TBS interaction was believed to be a major determinant for the specific binding of pre-tRNA (Loria et al. 1997) and the crystal structure has shown that this is indeed the case (Reiter et al. 2010).

The RCCA–/RNase P RNA motif interaction The RCCA is a preserved sequence in all species tRNA genes and it is lo-cated at the 3’end of tRNA. It is suggested to interact with the P15 loop of RNase P RNA (Kirsebom and Svärd 1994; Okabe 2003). The +74C and +75C pair with G293 and G292 in M1 RNA, respectively. These pairings are believed to be Watson-Crick base paring, which is one of the way ribozymes interact with their substrates. The other way of RNA-RNA association in ribozyme catalysis is through tertiary structure interaction and recognition, like TSL/ TBS interaction. Moreover it has also been suggested that U294 in M1 RNA interacts with +73 in the tRNA precursor (Kikovska et al. 2005; Brännvall et al. 2002, 2003, 2004). (see Figure 6) In the crystal structure published in 2010, the authors suggested that U294 and G+73 also form a base pair and the terminal A76 forms a weak interaction with G291 (Reiter et al. 2010). The RCCA-/RNase P RNA interaction is proposed to anchor the RNase P RNA on pre-tRNA, exposing the cleavage site and consequently making the cleavage occur on the correct site. Other interactions have been suggested in the vicinity of the RCCA interac-tion. A258 in the P-15 loop of M1 RNA, together with C75/G292 forms a triple base pair (Heide et al. 1999, 2001). Also in addition, A76 is suggested

24

to interact with G259 through an interaction with the Hoogsteen edge (Heide. 2001).

Figure 6. A diagram of the interactions in the vicinity of cleavage site (the big ar-row indicats the cleavage site). The RCC motif in tRNA base pairs with (U)GG in M1 RNA. C+74C+75 pair with G293G292 through WC-base pairing, but the U294/G+73 is not WC-base pairing. The -1U interacts with A248 from M1 RNA. The circled A, B and C show the position of functionally important divalent metal ions. The figure is modified from paper I.

The A248/ N-1 interaction The majority of tRNA precursors in E. coli have U at the -1 position (the residue right before the cleavage site). The A248 residue is suggested to interact with the -1 residue in pre-tRNA (Zahler et al. 2003) (see Figure 6). Data indicate that the interaction is perhaps not Watson-Crick base pairing. The two oxygens at position 2 and 4 on the base are suggested to play an important role (paper V), but in my study (paper IV and paper V), we found that A248 perhaps is not interacting with N-1 (for more discussion see be-low). However, cross-linking data indicate that several residues are posi-tioned close to the -1 residue. They are: A248, A249, C252, C253, G332 and A333. These residues might form a binding pocket for the -1 residue in the tRNA precursor (for review see Kirsebom, 2007). This interaction most like-ly works together with the RCCA/RNase P RNA interaction to help the RNase P bind correctly and thereby facilitate the efficiently cleavage.

U69/+5 interaction and 2’OH/RNase P RNA interaction The P4 helix (see Figure 5) has been suggested to be important for divalent metal ion (Mg2+) binding (Hardt et al. 1995; Harris et al. 1995; Christian 2000; Kaye 2002). These Mg2+ ions are important for the function of RNase P RNA. It is also suggested that residues in the vicinity of U69 from M1 RNA interact with the residue positioned 5 residues downstream of the clea-vage site in the tRNA (Christian et al. 2006). This interaction can affect the metal ion binding on the P4 and subsequently affects on the metal ion that is on the cleavage site.

Canonical cleavage site

294

tRNA +73

M1 RNA

M1 RNA

25

The 2’OH at the cleavage site is important for ground state binding, cleavage rate, cleavage site recognition, and metal ion binding in the vicinity. Toria et al. state that the 2’OH at the cleavage site mediates an interaction between RNase P RNA and its substrate (Toria et al. 1998). Others believe that it is the 2’OH that actually interacts with RNase P RNA (Brännvall et al. 2005, 2004; Zahler et al. 2005; Persson et al. 2003). For a schematic diagram of the different interactions, I refer to Paper I.

Different model substrates used in my study

Figure 7. Sketch of the secondary structure of different model substrates. The left substrate represents the full length tRNA precursor. The upper right substrate is the substrate that maintains T-stem/loop structure of the tRNAser precursor (the dotted line on the pTS-L substrate indicates the cutting site from where the pATSer sub-strates are generated ). pATSerUGGAAA is derived from pATSerUG substrate with an altered tetra T-loop. The lower right substrate does not have the T stem/loop struc-ture. The figure is modified from paper I.

In my study, I used different substrates to increase our understanding of the roles of various determinants in RNase P RNA-mediated catalysis. As de-scribed previously, RNase P RNA interacts with the tRNA precursor on the acceptor stem and D/T stem loop. Hence we used the pATSer series of sub-strates which have the acceptor stem and T-loop, and the T-stem from pre-tRNA to form a hairpin-like structure (McClain et al. 1987). This pATSer substrate therefore has the potential to interact with the TBS region. Our data suggested that the remainder T-loop of the model substrate does indeed in-teract with the TBS region in M1 RNA (see discussion below and Paper I). Because the TSL/TBS interaction is very important for RNase P RNA ca-talysis, we decided to generate another series of substrates. These were de-rived from the pATSer substrate but had 4 nucleotides to replace the T-loop while maintaining the 12 nucleotides long stem from pATSer. This substrate

pTS-L (U-1)

26

is called pATSerUGGAAA or pATSerCGGAAA, depending on the distinct resi-dues at the -1 and +73 positions. For example: UG equals to -1U/+73G, and CG is equivalent to -1C/+73G, etc. To be able to investigate the RNase P RNA-substrate interaction further, we further minimized the stem in the pATSerGAAA substrates to a 3-nucleotide pairing stem; this series of sub-strates is called pMini3bp substrate. (Figure 7) All these model substrates, i.e. pATSer, pATSerGAAA, and pMini3bp, can act as substrates of RNase P in vitro under both the physiological pH condition and a pH condition where the chemistry of cleavage is suggested to be rate limiting (Loria et al. 1998, Kikovska et al. 2005a, also see below).

A glance at the kinetics In my study all the kinetic constants were measured at pH 6.1, the pH at which the chemistry of cleavage is suggested to be the rate-limiting step. All the rate constants, except for some data in paper I, were determined under single turnover condition. This is a better condition than multiple turnover for measuring RNase P RNA mediated catalysis because the product-releasing step is the rate limiting step (See below).

Multiple turnover reaction

Figure 8. Scheme of a 3-step enzymatic reaction. E = free enzyme. S = substrate. P = product. ES = enzyme-substrate complex. EP = enzyme-product complex. k = rate constant.

The enzymatic kinetics can be measured in two different states: steady-state and transient state. The steady state of enzyme reaction means that the rate of enzyme-substrate (ES) formation equals to the rate of ES breakdown. The Michaelis-Menten equations are based on this state. When the amount of substrate (S) is more than that of the enzyme (E), each E has to make many “turnovers” to be able to cleave the substrates. This is

E+S k+1

k-1

k+3

k-3

k+2

k-2 ES EP E+P

kcat

kobs

27

called multiple turnover. In this condition Km, the Michaelis constant, can be written as shown here [Eq. 1]. Km is the concentration of substrate when the speed of reaction reaches half the maximum speed. If we draw a figure with [S] on the X-axis and rate on the Y-axis, it is not difficult to see that the smaller the Km, the stronger the interaction between enzyme and substrate. If the k2 is negligible, i.e., when k2 << k-1, then the Km can be written as shown in [Eq. 2]. In this situation, the Km can be considered as the dissociation constant of ES. kcat is the rate constant representing the speed of converting the ES to prod-uct (P). As Figure 8 indicates, kcat can be written like [Eq. 3 and Eq. 4]. kcat/Km is called the specific constant and it is an indicator of how well an enzyme catalyzes different substrates. When k2>>k1, then kcat/Km=k+1.

Single turnover reaction In an enzymatic reaction, the speed of the reaction is determined by the rate-limiting step. For example, if the product-releasing step is the rate limiting step, the entire rate of the reaction is dependent on this step, no matter how fast the other steps are. The limitation of studying steady-state kinetics in RNase P-mediated catalysis is that the limiting step under steady-state at saturated substrate concentration is tRNA product release, not pre-tRNA cleavage (Reich et al. 1988; Tallsjö et al. 1993). Therefore, studying the transient-state kinetics in RNase P RNA catalysis has a great advantage. This can also be called single turnover kinetics, which means the concentration of enzyme is much greater than that of the substrate. A single molecule of the enzyme can cleave at most one substrate molecule. Note that the protein part of the RNase P helps the product release, so the product releasing step might not be the rate-limiting step for the holo-enzyme. However the kinetics of the RNase P holoenzyme is still unclear.

ESSESE

kkk

K m)( 0

1

21 (1)

3232cat

1111 k

1kkkk

(3)

EVmax

catk (4)

1

1

kkKm (2)

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At pH 6.1, the chemistry of cleavage (k2) is suggested to be the rate limiting step in the RNase P RNA mediated reaction (Loria et al. 1997; Warnecke et al. 1996). This has been inferred from pH titration experiments. We use sin-gle turnover conditions at pH 6.1 in our reaction system for the analysis of RNase P RNA kinetics. Compared to multiple turnover condition, kobs/Ksto in single turnover condi-tions is equal to kcat/Km. kobs reflects the rate of cleavage of the scissile bond, which means kobs= k+2. According to the description above, kcat/Km =k+1, therefore kobs/Ksto = k+1. At pH 6.1, single turnover RNase P RNA mediated cleavage, we argue that if k-1>>k2, then Ksto Kd. (paper II, III and V). To investigate if the experiment is occurring at k-1>>k2 condition, a pulse-chase experiment (also called dilution experiment) was needed (see paper III). The key idea of this experiment was to dilute the already initiated reac-tion into a vast amount of reaction buffer (about 200 times in our experi-ment) to see if the reaction was “pulsed” due to the dilution. In our experi-ment we saw that without dilution the cleavage product increased with time. With dilution it did not (supplementary data in paper II, III and V). Thus, for RNase P RNA mediated cleavage at pH 6.1, single turnover condition, Ksto Kd.

Ground state binding and dissociation constant Kd is the dissociation constant. appKd stands for apparent Kd, which is also called ground-state binding constant (Kikovska et al. 2005)). This constant means that when the binding is strong between the two subunits, the appKd is small, and when the binding is weaker the appKd becomes bigger. For in-stance, the reaction for enzyme (E) binding the substrate (S) is carried as E•S E+S; the equation for this reaction is [Eq. 5]:

Hill coefficient Hill coefficient originally is used to quantify the effect of a ligand binding to a molecule. It expresses the degree of cooperativity of a ligand binding to an enzyme or molecule. In our research, we used the Hill coefficient to characterize the cooperativity of Mg2+ in RNase P RNA mediated cleavage. The number of Hill coefficient reflexes the number of functional important Mg2+ in the cleavage. We calcu-

ESSEKd (5)

29

lated the Hill coefficient from Mg2+ titration experiment. The Hill coefficient is defined by [Eq. 6] After rearrangement it can be written [Eq. 7]

In equation 6 and 7, v is the velocity under certain Mg2+ concentration. Vmax is the maximum velocity detected in a set of Mg2+ titration experiment, h is the Hill coefficient and a is the Mg2+ concentration that the v corresponds to.

)/(maxhhh akaVv (6)

hkahvVv loglog)/(log max (7)

30

About this thesis RNase P is essential in the cell and therefore understanding the mechanism of RNase P-mediated catalysis is important. It will help us to understand cleavage by ribozymes in general, the evolution of RNA and it will help us design new medical tools to cure, for example, diseases caused by different bacteria. Previous decades of studies have progressed our knowledge of RNase P structure, RNase P RNA folding, RNase P-substrate recognition and binding, the roles of divalent metal ions in RNase P RNA-mediated cleavage, the kinetics and mechanism of RNase P catalysis, and RNase P in the perspec-tive of evolution. However, there are still many unanswered questions that needs to be investigated. What is the basic chemistry of RNase P-induced cleavage? Which residues or chemical groups in the vicinity of the cleavage site contribute to the clea-vage? RNase P interacts with its substrate, but what is the interaction in the intermediate stage and how much do these interactions contribute to the ca-talysis? What is the conformation of the RNase P RNA-substrate in the tran-sition state and how does that conformation affect the cleavage? What is the exact role of divalent metal ions? What is the contribution of the S-domain and the C-domain of RNase P and what are their roles in an evolutionary perspective? What is the role of the proteins of the RNase P? In my study we used biochemical tools to answer some of these questions. Different constructs and mutants of RNase P RNA and model substrates were used in my studies to provide information. We compared data from Mg2+ profiles, cleavage site selection and cleavage rate of different RNase P RNA-substrate combination. From these data we rationalize the possible mechanisms in RNase P catalysis. In this thesis I will mainly focus on the RNA part of the RNase P. My work has contributed to the understanding of the RNase P RNA-substrate interac-tion, the function of the two domains of the M1 RNA, the evolution of RNase P and the role of the -1 residue in the mechanism of RNase P RNA mediated cleavage. In my study I used the M1 RNA and Pyrococcus furiosus (Pfu) RNase P, with and without the protein subunite, as model enzymes. Various tRNA-derived model substrate was used as the model substrates (See before).

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Induced fit model

There are several models in enzymatic catalysis to describe the procedure of how an enzyme interacts with its substrate, binds it, and eventually releases the product. The induced fit model, the lock and key model, the Concerted model and Sequential model are some of them. The lock and key model ex-plains the process as one where the enzyme and substrate form a complex from rigid complementary geometries, like a lock and a key. In contrast, the induced fit model states that the binding of the substrate slightly changes the conformation of the flexible active site of the enzyme and eventually leads to a better binding and catalysis (Figure 9). In the Concerted model and the Sequential model the structure of the enzyme changes after the binding of the substrate. In the Concerted model, the change is caused by the same sub-strate, but in the Sequential model the change of one subunit favors the change of another subunit.

Figure 9. Elucidation of the Induced fit model. The large, lower darker half circle indicates the enzyme and the lighter, upper form indicates the substrate. The color change of the substrate indicates the conversion of substrate to product. The figure is modified from Wikipedia (http://en.wikipedia.org/wiki/Induced_fit#Induced_fit).

The selection of the aminoacyl-tRNAs by the ribosome was suggested to follow the induced fit mechanism. Using kinetic studies, it was found that the codon-anticodon recognition not only stabilized the aminoacyl-tRNA binding on the ribosome, but also stimulated the forward reaction of the pro-

substrateActive site

Substrate is going to bind on the enzyme

Substrate binds to the Enzyme

The Enzyme-substrate com-plex formed

Products are released

The conformation of the active site changes slightly to fit the substrate

32

ductive pathway (Rodnina 2001). The hammerhead RNA is also suggested to use the induced fit mechanism. The Schistosoma mansoni hammerhead structure has a peripheral loop-bulge structure that can enhance the catalytic rate from a distal interaction (Nelson 2006; Martick and Scott 2006). An induced fit mechanism also has been suggested in RNase P mediated cleavage (Guerrier-Takada et al. 1989). In this study the residue number 92 was deleted in M1 RNA which resulted in a mutated M1 RNA with an al-tered cleavage site on the mutated substrate that lacked CCAUCA sequence at the 3’ end. This altered cleavage can be reversed by adding C5 (the pro-tein part of the E. coli RNase P) or 3M ammonium, which indicates that the structure of the enzyme-substrate complex is flexible.

Investigation in Paper I & Paper II Previous studies have shown that the TSL-region binds to the residues in the S-domain (TBS-region), and that changes in the TBS-region of B-type RNase P RNA affect substrate binding. Therefore, in paper I and II we wanted to investigate whether changes in the TBS-region in a type A RNase P RNA affect cleavage on the substrates that have various numbers of RNase P determinants. In paper I and II, our data suggest an induced fit mechanism in M1 RNA-mediated catalysis (paper I and paper II). We found that the productive TSL-/ TBS interaction can change the conformation of the M1 RNA-substrate complex and results in a distal influence on the events at the cleavage site. It is noteworthy that the effect is decreased at higher Mg2+ concentration. This emphasizes the importance of Mg2+ as well as the flexibility of this interac-tion. This observation has been further confirmed by experiments in paper II. In order to create a conformational change at the TBS region in M1 RNA, we used various mutations (or reverse) on the base pair G125/C235 that is located in the TBS region in M1 RNA. We used Pb2+-induced cleavage to confirm the conformational change in TBS region. As shown in the paper I, we obtained a change in the TBS region after we disrupted or reversed the G125/C235 base pair. When we increased the concentration of Mg2+ in Pb2+-induced cleavage, the cleavage pattern of mutant G235 was changed. This indicates that the base pair of G125/C235 is important for metal ion binding. In paper I and II, we used substrates with different structures and this al-lowed us to analyze the interactions between the TSL and TBS regions, as described above. To date, C-1/G+73 pairing leads to miscleavage at the -1 residue, which is one residue before the canonical cleavage site (Pettersson and Kirsebom 2008). It has also been suggested that a change at the -1 posi-tion disturbs the +73/294 interaction (see above). Therefore we used sub-

33

strates with mutations at the -1 residue to analyze the effect of the TSL/TBS interaction on the cleavage site. Note that all the alternative cleavage sites different from the canonical cleavage site, are consider as miscleavage. Because of the distinctive behavior of pATSerCGGAAA that we found in pa-per I, we decided in paper II to investigate the impact of the tetraloop GAAA. The tetra loop sequence GNRA that the model substrate pATSerCGGAAA has is well defined in structure (Heus et al. 1991). It is a stable construct and distinguishable from other tetra loop structures. pATSerCGUUCG and pAT-SerCGCUUG are two substrates derived from pATSerCGGAAA (see description above). These two tetraloop structures have been experimentally determined in the last decade (Jucker & Pardi 1995; Ennifa et al. 2000). We made these two tetra-loop substrates to define the effects of the tetra-loop structure in the cleavage site selection and rate of cleavage (see paper I, II III and IV; see above).

Cleavage site selection We observed in paper I and II that an altered TSL region in the substrates resulted in miscleavage. The pTS-L and pATSer substrates have the intact T-stem/loop structure, meaning that the TSL/TBS interaction is not disturbed, the M1 RNA and all the M1 RNA mutants can still cleave the substrates at the canonical cleavage site. But interestingly for pATSerCG: the -1 cleavage frequency went up as the Mg2+ concentration was raised. pATSerCGGAAA has a disrupted TBS/TSL interaction, therefore the M1 RNA cleaved this sub-strate mostly at the -1 position. In the case of pATSerUGGAAA, the TSL/TBS interaction has been disrupted, but the cleavage site is still exposed because U-1 cannot pair with G+73 (see above and Pettersson et al. 2008), that is most likely the reason why this substrate does not give miscleavage. Like pATSerCGGAAA, the other two tetraloop substrates all had miscleavage at -1 position (paper II), but the -1 cleavage frequency was much lower than for pATSerCGGAAA. The most interesting substrates are the pMini3bp substrates. They do not have the T-stem/loop at all. This means that the TSL/TBS inter-action is lost when these substrates are used. Therefore the impact of the TSL/TBS interaction on the cleavage site is lost as well. So as we observed in the experiment all the M1 RNA derivatives could still cut the pMini3bp substrates mostly at the right position, as in the case of the full length tRNA precursor. We also saw that changes in the TBS region of the M1 RNA rescue the mis-cleavage caused by all three tetra-loop substrates. This suggests that the TSL region (the T-loop structure) influences the cleavage site selection. This, and the comparison of the cleavage of M1 RNA with G235 on all tetraloop sub-strates and pATSerCG suggests that the topography in the vicinity of TBS region affected cleavage site selection as well.

34

Mg2+ profile To date, we have found that decreasing the size of the substrates results in an increase in the requirement of Mg2+. For example, the pMini3bp substrates required more Mg2+ than the others. From the Mg2+ curves we also calculated the Hill coefficient of the sub-strates in paper II (about the Hill coefficient see above). The Hill coefficient of pATSerCGGAAA was much higher compared to other tetraloop substrates and pATSerCG, especially at the -1 cleavage site. Apart from pATSerGAAA, the G235 mutant increased the Hill coefficient for the rest of the tetraloop substrates. Note that in paper III we found that the Pfu RNase P RNA (the TBS region is different compare to that in M1 RNA) gave the same Hill coefficient as G235 with tetraloop substrates. These data show: 1) The Mg2+ stabilizes the RNase P RNA-substrate com-plex, helping the RNase P RNA to cleave its substrates. 2)The lower the number of determinants, the higher the number of Mg2+ ions that are required for cleavage. 3) The loop structure affects the Mg2+ binding site and there-fore influence the RNase P RNA-substrate complex stabilization. 4) The structure of the T-loop affects the critical Mg2+ binding sites for +1 and -1 cleavage sites differently. 5) Changing the structure of the TBS region influ-ences the number of critical Mg2+ binding sites, and thereby influences the stability of the RNase P RNA-substrate complex.

Cleavage rate To further analyze the mechanism of catalysis, we determined the kobs and kobs/Ksto for all the substrates under single turn over and pH 6.1 conditions (see above about the kinetics). To date, we found: 1) The fewer the number of determinants that the substrate has, the less efficiently the M1 RNA cleaves it. The pMini3bp substrates had the lowest kobs; pTS-L and pATSer were the fastest substrates. 2) The C-1 substrates had lower efficiency com-pare to the U-1 substrates. This might be due to the exposure of the cleavage site. In other words, if the cleavage site is open (as is the case in the U-

1/G+73), the M1 RNA cleaves more efficiently. For example, pATSerUG is a faster substrate than pATSerCG. 3) Productive TSL/TBS interaction influ-ences the rate of cleavage. This was seen in the cases of pTS-L and pATSer derivative substrates that were cleaved by the M1 RNA mutants - the rates of cleavage decreased. In the case of pMini3bp substrates, however, the mu-tants of the M1 RNA did not lower the efficiency of cleavage. This is the same as when pATSerGAAA substrates were used. 4) The loop conformation influenced the cleavage rate of RNase P RNA at the cleavage site. The pAT-SerCGUUCG and pATSerCGCUUG were much slower substrates for M1 RNA compared to pATSerCG and pATSerCGGAAA. 5) Changes in the TBS region

35

could also influence the cleavage rate and binding affinity. This was seen by comparing M1 RNA to G235 cleavage of the tetraloop substrates. Note that in paper II we used the optimal amount of Mg2+ in determining the rate of cleavage. This was not the case in paper I. For example in paper I we observed that pTS-L(U-1) and pATSerUG had different rates at low Mg2+ concentration, but the difference became much less when the Mg2+ concen-tration increased. This is probably because we did not use the optimal amount of Mg2+ in those experiments. The optimal (approximately) amount of Mg2+ (according to the Mg2+ profiles that we had for all the RNase P RNA- substrate combinations) allowed us to compare the results in all the RNase P RNA-substrate combinations without a risk of bias caused by the different Mg2+ requirements (see above and paper I & II).

Binding constant The binding constant appKd

differed when the structure of the substrate or the -1 residue identity changed. To date, disturbing the TSL/TBS interaction increases appKd. However, for the substrates that lack the interaction with the TBS region, structural changes of the TBS region do not make signifi-cant differences on the binding constant. Moreover, changing the -1 residue from U to C also increased the binding constant, but these increases were reduced when the divalent metal ion concentration was increased. This shows the importance of the divalent metal ion in the RNase P RNA medi-ated catalysis. For further information, I refer to Kikovska et al. 2006, 2005; Zahler et al. 2003; Loria et al. 1997.

Mn2+ and cleavage site selection Mn2+ can also induce RNase P RNA cleavage (Brännvall et al. 1999). It can cause miscleavage at -1 when pATSerCG is cleaved by the M1 RNA wild type. However, in our experiments we observed that the G235 mutant can rescue the miscleavage. This strongly indicates that a change in the structure topography of TBS region influences the position of divalent metal ion(s) in the vicinity of the cleavage site. This might also be one of the reasons why the miscleavage was recovered when the structure of the TBS region was changed.

Alterations in and at the vicinity of the cleavage site Previous data have shown that changing U to C at position 294 in M1 RNA (the U294/ +73G interaction, part of the RCCA/ P15 interaction, Fig. 6) could make the effect of the C-1/G+73 disruption smaller (Brännvall 1999; 2001). In my experiments I found that C294 actually rescued, to some extent, the miscleavage caused by the M1 RNA wild type, especially for tetraloop substrates. C294 also influenced the Hill coefficient of pATSerCG. When it comes to the rate of cleavage, C294 did not increase the rate of pATSerCG

36

much, but C294 increased the rate of pATSerCG tetraloop substrates at the +1 cleavage site, but not on the -1. However the binding constant was quite similar for the M1 RNA wild type and G235 when tetraloop substrates were used. In conclusion, the data here show 1) Strengthening the +73/294 inter-action compensate for a nonproductive TSL/TBS interaction to some extent. 2) The mutation from U to C at position 294 might affect functionally criti-cal Mg2+ binding at the vicinity of the cleavage site. 3) Strengthening the +73/294 interaction also improves the cleavage rate, especially at the +1 cleavage site. 4). C294 works like a suppressor for cleavage at the -1 site. We also exchanged the 2’OH to 2’H on residue G+1 in the pATSerCGGAAA which resulted in lower cleavage frequency on -1 compare to intact pAT-SerCGGAAA, when cleaved by M1 RNA variants. In summary The productive interaction of TSL/TBS influences the cleavage site selec-tion, RNA-substrates binding, Mg2+ requirement and the rate of cleavage. The evidence indicated that M1 RNA-mediated cleavage can be explained by the induced fit mechanism. The divalent metal ions, together with the chemical groups in the vicinity of the cleavage site, also play an important role. The identity of the -1 residue also contributes to the efficiency of cleav-age. G235 is important in the TSL/TBS interaction. The induced fit model in paper I and II is the first properly investigated such kind of mechanism in RNase P RNA-mediated catalysis. The RNase P RNA-substrate-protein complex crystal structure (Reiter et al. 2010) pro-vided new data that allowed us to analyze the Induced fit model in more detail and get a further understanding of the mechanism. From a close ex-amination of the residues in contact with the TSL region, we suggest that stacking might be the important mechanism for establishing a productive TSL/TBS interaction (see paper II). Whether this is also the case for other types of RNase P RNA remains to be discovered.

37

The two domains of the RNase P RNA

RNase P RNA has two domains, as mentioned before (Figure 5). To date, the two domains of RNase P RNA fold to their native structure independ-ently of each other (Baird NJ et al. 2005). However, data also showed that the two domains interact with each other during folding in type A RNase P RNA. The L8 loop and P4 helix interact with each other and the L9 loop interacts with the P1 helix (Massire et al. 1998). That says that domain-domain interactions play an important role in the folding of type A RNase P RNA. These two domains are not only folding independently of each other as well as cooperatively, they also contribute to the function of RNase P RNA both independently and cooperatively.

Contribution of the S-domain We chose Pyrococcus furiosus (Pfu) RNase P RNA in paper III to investi-gate the impact of the S-domain and the relation between S-domain and C-domain. Pyrococcus furiosus (Pfu) is an Archaea species. This species is special because the optimal growth temperature for it is 100°C. At this tem-perature most of the living organisms would be destroyed. Thus Pfu is classi-fied as a hyperthermophile. Archaeal RNase P contains one RNA subunit and 4-5 proteins. The RNA subunit has the catalytic property on its own in vitro (Pannucci et al. 1999; Tsai et al. 2006; Li et al. 2008).The RNA subunit of Pfu RNase P belongs to the A-type RNase P RNA, the same type as E. coli RNase P RNA. A com-parison of the secondary structures of these two RNAs shows that they have similar C-domain but different S-domain structure in the P10 and P11, where the TBS region is located (Gopalan, 2007 and description above). The spe-cial properties of Pfu RNase P are that its protein part is homologous to the eukaryotic RNase P protein (Hall and Brown 2002), but the RNA part is more like bacterial RNase P RNA that performs catalysis in vitro on its own at a much higher efficiency than Human’ RNase P RNA (Pannucci et al. 1999; Tsai et al. 2003, 2006).

38

The Pfu holoenzyme has been successfully re-assembled in vitro by Gopa-lan‘s group (Tsai et al. 2006), providing us with an opportunity to study the Pfu holoenzyme part by part in vitro.

Present investigation in Paper III We used Pfu RNase P, both with and without the protein part, to cleave our model substrates. Pfu RNase P could recognize these substrates and cleave them in vitro, both in the absence and presence of the protein part. As mentioned previously, Pfu RNase P RNA has a similar C-domain as E. coli RNase P RNA (M1 RNA), but the TBS region in its S-domain is differ-ent. M1 RNA cleaved the substrate that has a paired cleavage site and a dis-turbed TSL region mainly on -1, i.e., pATSerCGGAAA. If the TBS region in the M1 RNA was disrupted, then the miscleavage was recovered. However, in the Pfu RNase P RNA, the TBS region is different from M1 RNA, and the substrate pATSerCGGAAA was cut mainly at +1 instead. However, if we exchanged the S-domain of Pfu with the S-domain of M1 RNA, the newly constructed RPREcS3 cleaved pATSerCGGAAA again mainly at -1, just like we observed when we used M1 RNA to cleave this substrate. This interesting observation indicates the importance of the S-domain, espe-cially the TBS region, in the cleavage site selection. The S-domain of Pfu RPR was suggested to interact differently with the substrate compared to M1 RNA’s S-domain. This also follows the induced fit model that we proposed in the paper I and II: a change of the TBS/TSL interaction can lead to con-formational changes of the RNase P RNA-substrate complex and from distal disturb the happening at the cleavage site. A similar observation was made by replacing Methanothermobacter thermautotrophicus RNase P RNA’s S-domain with M1 RNA’s S-domain. This resulted in an improvement in the catalytic activity (Li, 2008). The improvement might also be due to im-proved substrate binding by using the M1 RNA’s S-domain. The interesting aspect of the Mg2+ profile is that pATSerUG required more Mg2+ when it was cleaved alone by Pfu RPR, compared to when the M1 RNA was used. The pATSerUG and pMini3bpUG had very similar Mg2+ requirements, which was the opposite for to M1 RNA induced cleavage. Pfu is a hyperthermophile, so we determined the cleavage rate of Pfu RNase P RNA on the model hairpin substrate both at 37 C and 55 C (55 C is the optimal cleavage temperature for Pfu RNase P RNA). Pfu RNase P RNA is a slower enzyme compared to M1 RNA. Pfu RNase P RNA gives faster cleav-age at 55 C, which is consistent with the observation that was made with for Thermus therimophilus RNase P RNA (Hartmann, 1991). There was almost

39

no difference in rate and dissociation constant Kd for pATSerUG compared to pMini3bpUG when cleaved by Pfu RNase P RNA. The protein subunit of Pfu consists of 4 proteins that form two binary com-plexes: POP5•RPP30 and RPP21•RPP29. The footprint experiment showed that the RPP21•RPP29 binary complex might affect the TSL/TBS interaction or it might actually bind to the TSL region of the substrate (Xu, et al. 2009). Our experiments showed that when Pfu RNase P RNA cleaved the substrate in the presence of RPP21•RPP29 at a lower Mg2+ concentration, it behaved like the wildtype M1 RNA. However, when the Mg2+ concentration in-creased, the miscleavage was recovered, like the case with only Pfu RNase P RNA. The POP5•RPP30 was suggested to bind on the C domain of Pfu RPR (Tsai, et al. 2006; Xu, et al. 2009). In contrast to RPP21•RPP29, POP5•RPP30 did not make any significant change on Pfu RNase P RNA cleavage site selection. The holoezyme behaved in the same way as RPP21•RPP29+ Pfu RNase P RNA with respect to cleavage site selection. All these data indicate that 1) The TSL/TBS interaction is important for cleavage site selection and the RPP21•RPP29 significantly influences on the cleavage selection. 2) Mg2+ plays an important role in the interaction. 3) This is further support for previous investigation that found that the C-1/G+73 pair-ing in pATSerCGGAAA influences the cleavage site selection. In summary 1) The S-domain was important for substrate binding, cleavage rate and cleavage site selection (also see Brännvall et al. 2007). 2) The S-domain from Archaeal RNase P RNA interacted differently with the substrate com-pare to the bacteria one. 3) The Pfu RNase P protein complex RPP21•RPP29 could alter this difference. 4) The TBS/TSL interaction was important for cleavage by disturbance from distal of the cleavage site conformation or by disturbance of the metal ions or chemical groups in the vicinity of the cleav-age site. 5) The C-1/ G+73 could lead to miscleavage. 6) POP5•RPP30 inter-acted with the C-domain of the Pfu RPR and only led to miscleavage on the substrates that had only RCCA-/RNase P RNA interactions. 7) The position-ing of the divalent metal ion was very important for the cleavage. The data suggested that changing the positioning of the divalent metal ion led to low efficiency cleavage and incorrect cleavage.

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Contribution of the C-domain The catalytic domain (C-domain) is considered the ancient part of the RNase P RNA (Sun, et al. 2010). The C-domain can perform catalytic activity in vitro without the S-domain, in the absence and presence of the protein part (Green and Vold, 1996; Loria and Pan, 1999; Kikovska et al. 2011). There-fore the C-domain might have existed first as a catalytic RNA. Then, as re-quirements evolved, like higher rate and complexity in substrate selection, the S-domain evolved, followed by the evolution of the protein part of RNase P. There exist small RNase P RNAs in nature which resemble the secondary structure of the C-domain. For example, the Saccharomyces cere-visiae mitochondrial RNase P RNA does not have the corresponding S-domain nor the P15-17 region (Seif et al. 2003), but this RNase P RNA has no catalytic activity without the protein part of the RNase P RNA in vitro. However, there are two other mini RNase P RNAs from Caldivirga ma-quilingensis and Vulcanisaeta distribute (Lai, et al. 2010). These two mini RNase P RNAs lack the structure corresponding to the specificity domain and they can actually act in trans, in vitro on the substrates without the help of the protein. The existence of these small RNase P RNAs in nature pro-vides evidence that the C-domain most likely was the first catalytic part of RNase P RNA that evolved in nature.

Present investigation in Paper IV In paper IV we wanted to investigate the impact of the C-domain in catalysis by RNase P RNA. In our experiments we observed the catalytic activity in vitro of the C-domain construct (CP RPR) on the model substrates (see paper II and IV and descriptions above). The CP RPR cleaved all the tetraloop substrates at the canonical cleavage site irrespective of the concentration of Mg2+, which is in contrast to our observations using M1 RNA (see paper I, II). This was expected because the C-domain does not have the TSL/TBS interaction with the substrates, as in the case of M1 RNA cleavage of pMini3bp substrates (see paper I and IV). The S-domain therefore influences the cleavage site selection. Without the TBS region interacting with the TSL region, the RNase P RNA generates much less miscleavage on its substrate. This again provides a link between TSL-/TBS and events at the cleavage site. Looking at the Mg2+ profile, we observed that there was almost no difference in the Mg2+ requirement for pATSerUG and pMini3bpUG when CP RPR was used, which is in contrast to M1 RNA (see paper I). We also calculated the Hill coefficient. Table 2 shows that pMini3bpUG required more Mg2+ than pATSerUG during the cleavage of M1 RNA. We observed the same

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effect when pATSerCG and pATSerCGGAAA were cleaved by CP RPR and M1 RNA: they both need higher amounts of Mg2+ when cleaved by CP RPR, but there was no difference between them when cleaved by CP RPR (Table 2, taken from Wu et al. manuscript). These observations also reflect the impor-tance of the TSL/TBS interaction. When the S-domain is deleted, more Mg2+ will be needed to stabilize the RNase P RNA-substrate complex. The base pairing of C-1/G+73 also increases the Mg2+ requirement. Earlier studies have suggested that Mn2+ can coordinate differently in the vicinity of the cleavage site resulting in increased miscleavage frequency at the -1 position (Brännvall and Kirsebom 1999; 2001; Brännvall et al. 2002, 2003 and 2007; Kirsebom 2007). However, when CP RPR was used, the miscleavage at -1 did not increase, suggesting that the TSL/TBS interaction influences the positioning of the functional divalent metal ions at and in the vicinity of the cleavage site.

Table 2. Hill coefficients of different substrates with M1 RNA and C-domain

M1 RNA CP RPR

pATSerUG +1 2.1 ± 0.023 3.3 ± 0.5 pMini3bpUG +1 3.2 ± 0.19 3.2 ± 0.33 pATSerCG +1 3.4 ± 0.46 2.4 ± 0.1 -1 1.4 ± 0.39 pATSerCGGAAA +1 3.7 ± 0.37 2.8 ± 0.3 -1 4.6 ± 0.24

CP RPR gave a much lower rate of cleavage compared to a full-length RNase P RNA. The dissociation constant Kd also dropped when the C-domain was used compared to M1 RNA. However there were no significant differences between pATSerUG and pMini3bpUG in the rate and Kd value when CP RPR was used, which is different from M1 RNA-mediated cleav-age. The similar effect was seen with pATSerCG and pATSerCGGAAA. The data above suggest that the presence of the S-domain increases the binding affinity and rate of cleavage. Nonethelss, when the C-1/G+73 kind of sub-strates were cleaved by CP RPR, they gave significantly reduced rates com-pared to M1 RNA. A similar phenomenon has been seen in type B RNase P RNA mediated cleavage as well (Loria and Pan, 1999). These observations bring up an interesting interpretation that the result of the evolution of the S-domain is to increase the rate of cleavage, in particular by suppressing the negative influence of the C-1/G+73 base pair on the cleavage efficiency. At the same time the accuracy of cleavage is lost.

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In our experiments we found that changing the U294 to C294 in M1 RNA resulted in a rate increase when pATSerCG and pATSerCGGAAA were used (paper II). This was observed in CP RPRC294 induced cleavage as well, but the rate increase of tetraloop substrate cleavage with CP RPRC294 is not as great as during the cleavage with the full-size M1 RNA. This result suggests that the +73/294 interaction plays a role in cleavage efficiency. It also indi-cates that TSL/TBS interaction might contribute to breaking the C-1/G+73 pair in the substrate when the RNase P RNA-substrate complex is established (also see paper I and II). When we compared the substitution of A248 with G248 in M1 RNA with CP RPR mediated cleavage, we observed something different. The M1 RNAG248 cleaved pATSerCG at an increased rate at -1 compared to wild type M1 RNA, while the rate at +1 remained unchanged. The Hill coefficient changed slightly for M1 RNA wild type and M1 RNAG248. We also observed that the M1 RNAG248 binds pATSerCG with lower affinity compared to M1 RNA wildtype. This was not the case when CP RPR was used in cleaving pATSerCG. The rate of cleavage by CP RPRG248 actually increased signifi-cantly compared to wildtype CP RPR as did the Hill coefficient when CP RPRwt was mutated to CP RPRG248. This could indicate that the N-1/G248 in-teraction influenced the charge distribution in the vicinity of the cleavage site. This in turn affected the positioning of the divalent metal ion and as a result, the cleavage rate for CP RPRG248 increased compare to CP RPRwt. These data together emphasize the coupling of the TSL/TBS interaction and the events in the vicinity of the cleavage site. In summary 1) Deleting the S-domain resulted in less efficient cleavage. 2) Absence of the S-domain resulted in higher accuracy in the cleavage. 3) The structural architecture of the -1/+73 pair is important for the cleavage, for both the rate and the cleavage site selection. 4) A productive TSL/TBS interaction can help to open the C-1/G+73 by influencing the N-1/A248 and +73/294 interac-tions. 5) The results in this paper support the induced fit model.

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What is happening around the cleavage site?

RNase P cleaves its substrates at the canonical cleavage site. Studies around the cleavage site have always been one of the big focuses in RNase P re-search. Those studies include: the interactions in the vicinity of the cleavage site; the residues in the vicinity or at the cleavage site and chemical groups and critical Mg2+ binding sites around the cleavage site. There are a number of interactions, between RNase P RNA and pre-tRNA near or at the cleavage site: the RCCA-/ P15 loop interaction; the -1/A248 interaction; and the +73/U294 interaction (see introduction above). The pro-tein part of bacterial RNase P was suggested to interact with the 5’ leader sequence of the pre-tRNA (Niranjanakumari et al. 1998). The -1/ A248 inter-action was reported by Zahler and colleagues in 2003. They found that mu-tating A248 can lead to miscleavage of substrates that have 2’H at N-1. Spe-cifically, they showed that U248 can lead to a miscleavage of the pre-tRNA when the -1 reside is deoxy U, but that this miscleavage can be restored if the -1 residue is deoxyA. The same observation was also observed on the RNase P holoenzyme reaction. It was shown that the A248 binding defect can also be recovered in this way. They proposed that the U-1/A248 interac-tion is common and important for RNase P mediated catalysis (Zahler et al. 2003). However our data are not consistent with the idea that A248 interact with residue -1 through WC base pairing (see below). The C+1/G+72 base pairing in the tRNA precursor cleavage site is believed to be important for the binding and rate of RNase P RNA-mediated cleavage (Kikovska et al. 2005). Ema Kikovska and colleagues suggested that the exocyclic amine (2NH2) of G+1 affects the rate of cleavage. Absence of the 2NH2 affects the charge distribution and positioning of the Mg2+ binding site in the vicinity of the cleavage site. They also found that the base of the +1 residue is not absolutely necessary, although its presence suppresses the miscleavage and significantly increases the rate of the cleavage (Kikovska et al. 2006). The -2 residue in the tRNA precursor has also been suggested to play a role in both the cleavage site recognition and the rate of cleavage (Brännvall et al. 1998). In Brännvall’s work, they used M1 RNA and Mycoplasma hyopneu-moniae (Hyo P) RNase P RNA, a type B RNase P RNA. They found that

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these two RNase P RNAs behaved differently when the identity of the -2 residue changed in the tRNA precursor. The contribution of the -1 residue in the substrate About 67% percent of the -1 residue in E. coli tRNA precursors are uridines (Yuriko et al 1990, Kufel and Kirsebom 1996). If the -1 residue is C and can pair with G+73, this most likely leads to miscleavage at the -1 position, both in vitro and in vivo (Pettersson and Kirsebom 2008). Data in paper I also showed that C-1/G+73 substrates need more Mg2+ than U-1/G+73 substrates to reach the optimal cleavage. In addition, C-1/G+73 substrates are usually slow-er than U-1/G+73. Previous work has established the importance of the -1 residue in the tRNA precursor. The cleavage site selection and kinetics of cleavage are dependent on the identity of N-1 (Brännvall et al. 2002; Zuleeg et al. 2001). Brännvall and colleagues found that it is important that the -1 residue is a U nucleotide (it is also the most common -1 residue in the tRNA precursor), indicating that it is a positive determinant for RNase P cleavage. On the other hand, Zuleeg and colleagues used a mini model substrate and they mainly deter-mined the rate of cleavage. Changing the N-1 residue in a full-length pre-tRNA also leads to miscleavage and a change in the the rate of catalysis by the RNase P holoenzyme (Crary et al. 1998; Loria et al. 1998; Loria and Pan 1998). The 2’OH at the -1 residue is also important for the rate of cleavage (Smith and Pace 1993; Loria and Pan 1998, 1999). Pan and colleagues showed that a 2’H mutant influenced the rate of cleavage on both correct and incorrect sites. Brännvall and others also made a change on the -1 residue by replacing the 2’OH with 2’NH2. They found that the 2’N at -1 influenced cleavage-site recognition, substrate binding, and rate of cleavage. They also suggested that the 2’OH of the -1 residue is important for the charge distribution in the vi-cinity of the cleavage site, as the result influences the positioning of the di-valent metal ion (Brännvall et al. 2003, 2004; Persson et al. 2003).

Present investigation in Paper V For all the reasons above, we decided to investigate the impact of residue -1 in the tRNA precursor. In particular, we wondered why U-1 makes a faster substrate than the other variants in E. coli. C-1/G+73 substrates were slower than U-1/G+73 in our experiments with the model hairpin structure substrates, but not with the full tRNA precursor (Zahler et al. 2003; paper I). This raises the possibility that with an increas-

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ing number of determinants, the difference of C-1/G+73 and U-1/G+73 can be masked. To investigate the impact of the -1 residue, we decided to use the pMini3bp variant substrates that only carry the determinants around the cleavage site. This means the influence from the TSL/TBS interaction is removed (see above). We made a substrate based on the pMini3bp substrate structure but without the base attached on the ribose at the -1 residue; we called this pMini3bp /G. This substrate could be cleaved by M1 RNA in our experiment, but it gave about 50% miscleavage on the -1 residue. The Mg2+ profile of pMini3bp /G was almost the same as that of pMini3bpUG, but the rate of cleavage dropped dramatically when the base was deleted. All these data indicate that the base on the -1 residue is not absolutely needed for cleavage, but its ab-sence leads to miscleavage and serious drop in rate. As mentioned before, U-1/G+73 is a faster substrate than C-1/G+73. To avoid the impact of C-1/G+73 pairing, we also compared pMini3bpUA and pMini3bpCA substrates. Data showed that both substrates were almost al-ways cleaved only at the canonical cleavage site; pMini3bpUA was faster under single turn over condition compared to pMini3bpCA, regardless of the Mg2+ concentration. This result is in agreement with the multiple turnover measurement that was obtained by Brännvall (Brännvall et al. 2003). The results above indicate that U at the -1 residue makes a faster substrate than C. O4 in the base of U-1 contributes to the cleavage, but not significantly. (see Figure 10a) To further investigate the O2 in the base of U-1, we made the pMi-ni3bpIsoCG substrate. As Figure 10a shows, IsoC is derived from a cytosine. The difference between IsoC and U is that U has an oxygen at position 2, but IsoC has an NH2 group at this position. pMini3bpIsoCG could be cleaved by M1 RNA and usually only at +1 cleavage site, regardless of the concentra-tion of Mg2+. The Mg2+ profile of this substrate was the same as for the other pMini3bp substrates, but the rate of cleavage was much lower compared to pMini3bpUG. The binding constant, however, increased somewhat com-pared to pMini3bpUG. These results indicate that the O2 contributes signifi-cantly to the cleavage and should be considered to be a positive determinant for the M1 RNA mediated catalysis.

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Figure 10. Different base structures used in our experiments. a). Shows the differ-ence of C and U and Iso C. The numbers on the base ring indicate the base number-ing. b). Shows the different varieties of purine. The arrows show the difference of the NH2 group on the molecular structure and the effect that we observed in our experiments.

In E. coli, about 13% of tRNAs have A as the -1 residue and 8% have G as -1. Therefore, we wanted to investigate the effect of purine as a base at the -1 residue. We used 6 different pMini3bp substrates with purine base at -1 posi-tion for cleavage of M1 RNA (See Figure 10b). These substrates all needed high concentrations of Mg2+ for the cleavage and the rates were much slower

a

1

2

3 4

5

6

b

DAP

2DAP

Inosine Purine

Adenine

Guanine

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than for substrates with pyrimidine at -1. As Figure 10b shows, we com-pared the rate of different purine substrates. The data indicate that NH2 at position 2 on the purine base has a negative impact on rate, but no effect on the binding constant. The NH2 at position 6 does not affect the rate. pMi-ni3bpGG, pMini3bp2APG and pMini3bpDAPG gave miscleavage, but not the other substrates. These data indicate that NH2 at position 6 does not have much effect on cleavage selection. We also used the protein part of the E. coli RNase P (C5) to investigate the impact of C5 on the cleavage. To date, with help of C5, pMini3bpGG abol-ished the miscleavage. This implies that C5 acts as a positive determinant that can rescue the miscleavage caused by the 2NH2. It is worth noting that G-1 is not pairing with G+73 in the substrate that we were using. In previous studies, U-1 and A248 were believed to form a base pair with each other (Zahler et al. 2003). In the RNase P-substrate co-crystal structure A248 is located close to the 5’ termini of the tRNA (Reiter et al., 2010). However, we still cannot conclude if U-1 and A248 interact with each other because the crystal structure represents the post cleavage state, not the transi-tion state interaction. To investigate if the U-1 is forming a WC base pair with A248, an internal modification has been made on position 3 of the base U on -1 residue (Brännvall and Kirsebom, 2005). To date, together with the fact that C-1/G+73 has increased miscleavage frequency when A248 is changed to G248, we conclude that most likely U-1 and A248 do not interact via WC base pairing. It has also been suggested that the Hoogsteen surface of A248 is important in the interaction with the substrate (Siew et al., 1999). However, our experiments show that this is still unlikely because the cleav-age rate did not increase when we replaced the A248 with G248 on cleavage of -1pMini3bpIsoC (Figure 11). This result indicates that -1U is not base pairing with A248. The Mg2+ stabilizes the RNase P RNA-substrate complex and the O2 (and NH2 in G-1) in the base of the -1 residue perhaps involved in Mg2+ binding. This, together with the Mg2+ profiles, lid us to propose that the identity of residues or chemical groups in the vicinity of the cleavage site influences the charge distribution and thereby disturbs the positioning of the divalent metal ion, consequently influencing the rate and accuracy of cleav-age. This model is also supported by Kikovska’s result (Kikovska et al. 2006). In conclusion, in paper V we found: 1) The presence of the base in the -1 is not essential for M1 RNA mediated cleavage. 2) The two oxygens in the base of U-1 contribute to the cleavage; in particular, O2 contributes the most to the catalysis. 3) Purines as bases at -1 residue promote in poor cleavage compare to pyrimidine. 4) 2NH2 on the purine base is a negative determi-nant, both for cleavage accuracy, and efficiency of cleavage at the canonical cleavage site. 5) Residue at -1 is likely not involved in WC basepairing with

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A248. However, the identity of the residue in the vicinity of the cleavage site influences the charge distribution and thereby influences the position of the functionally important Mg2+.

Figure 11. Schematic diagram of possible -1U/A248 interaction through Hoogsteen edge. The dark circle indicates the residues that are involved in the Hoogsteen edge- interaction.

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Concluding remarks

The work that I present in this thesis contributes in understanding according to the following: 1) The importance of the base in the -1 residue in M1 RNA mediated cleav-age. The base in the -1 residue is not absolutely necessary, but its absence causes miscleavage on the -1 cleavage site. The O2 in the Uracil base con-tribute positively to the cleavage rate. The purines are not good for RNase P RNA mediated cleavage. 2NH2 influences the cleavage negatively. The -1 residue is not pairing with A248. The identity of the -1 residue influences the charge distribution, therefore influences the Mg2+ positioning and the cleav-age. 2) Functional coupling between the TSL/TBS interaction and the events at the cleavage site. The TSL/TBS interaction affects the cleavage site selection, cleavage rate and ground state binding. It can also affect the position of Mg2+ in or at the vicinity of cleavage site. G235 in M1 RNA is important in the TSL/TBS interaction. C-1/G+73 pairing affects the cleavage site selection. Finally we suggest that stacking plays an important role in RNase P RNA-substrate interaction. 3) Contribution of the S-domain to the cleavage. The S-domain is important for the cleavage rate, the cleavage site selection and the substrate binding. It increases the cleavage rate but decreases the accuracy of cleavage site selec-tion. The S-domain of Pfu RPR interacts differently from that of M1 RNA. Constructs that contain E. coli S-domain and Pfu C-domain have a cleavage site selection similar to that of E. coli RNase P RNA (M1 RNA). Pfu RPP21•RPP29 binds on the S-domain of Pfu RNase P RNA and confers the Pfu RNase P RNA with a cleavage site selection similar to M1 RNA. The POP5•RPP30 binds on the C-domain of Pfu RNase P RNA and leads to mis-cleavage when the only determinants are in vicinity of the cleavage site. 4) Contribution of the C-domain. Without the S-domain, C-domain gives more accurate, but slower, cleavage. This is important for understanding the evolution of the RNase P RNA. Without the TSL/TBS interaction, there is a higher requirement of Mg2+ in substrate-enzyme stabilization. The +73/294 interaction is important for cleavage efficiency. The N-1/248 interaction in-fluences the positioning of the divalent metal ion and cleavage efficiency.

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G248 especially influences the charge distribution and results in reposition-ing of the divalent metal ion. In our study we compared the Mg2+ requirement, cleavage site selection, and cleavage rate of different substrate-enzyme combinations to study the me-chanism of RNase P mediated cleavage. The RNase P-substrate cocrystal structure solved in 2010 (Reiter. et al. 2010) has contributed significantly to the research on RNase P. The combination of biochemical evidence and direct evidence, perhaps from evolving methods in biochemistry and struc-tural biology will provide us with more information to eventually solve the mechanism of RNase P mediated cleavage. For example, in studying the transition state cleavage, the developing non-crystal-dependent femtosecond X-ray method may be useful. One of the most important tasks of biology is to cure disease and prolong human life. To this end we are trying to find drugs that can target disease but do not damage the cell in the process. RNase P is a perfect potential drug target, for two reasons. First, RNase P is indispensable for bacterial viability (Schedl et al. 1974; Wegscheid and Hartmann 2006; Wegscheid et al. 2006; Kobayashi et al 2003). Second, the architecture of RNase P differs in Bacte-ria and Eukarya. For example, the loop (p15 loop) that is missing in Eukarya has a significant role for Bacteria, making it a good target for drug design. There are several approaches that have been developed to design drugs tar-geting RNase P. For example aminoglycosides has been found to inhibit the catalytic activity of RNase P. Hopefully some of the knowledge we get today can be used as tools to solve health problems in the future.

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Swedish Summary

RNas P är ett ribonukleas som klyver bort den extrasekvens som finns i 5’-änden av omogna tRNA-molekyler. RNas P är essentiellt och har hittats i alla levande organismer. Det består (med enstaka undantag) av minst en RNA-komponent (RPR) och en proteinkomponent (RPP). Antalet protein-komponenter varierar beroende på ursprunget hos RNas P. I bakterier finns endast ett RNas P-protein, medan det finns fyra till fem olika proteiner i arkéer och minst nio hos människor. Det är RNA-komponenten som står för den katalytiska aktiviteten (förutom i ett fåtal fall där RNas P enbart består av protein) och kan klyva omogna tRNAn in vitro (i provröret) i frånvaro av proteinkomponenten. Därför är RNas P ett ribozym. Inuti levande organis-mer, in vivo, är dock proteinkomponenterna livsnödvändiga. Eftersom RNas P är absolut nödvändig i cellen är det av vikt att förstå me-kanismen för RNas P-medierad katalys. Detta kommer att leda till en allmän förståelse av ribozymkatalyserad klyvning och evolution av RNA-molekyler. Dessutom kan kunskap om RNas P leda till att vi kan utveckla nya läkeme-del mot exempelvis olika bakterierelaterade sjukdomar. Under tidigare årti-onden har framsteg gjorts som gett oss förståelse för hur RNas P:s struktur ser ut, för hur den viker ihop sig från en endimensionell sekvens till sin slut-liga tredimensionella form, för hur RNas P känner igen och binder till sina substrat, för vilken betydelse tvåvärda metalljoner har för klyvningen, för vilken mekanism och vilken kinetik som kännetecknar katalysen, och för hur RNas P har utvecklats sett från ett evolutionärt perspektiv. Trots att mycket redan är känt finns flera obesvarade frågor som behöver lösas. Hur ser den grundläggande kemin bakom RNas P-medierad katalys ut? Vilka nukleotider eller kemiska grupper i det katalytiska sätets närhet är inblandade i klyv-ningsprocessen? RNas P interagerar med sina substrat, men hur ser interak-tionen ut mellan det att substratet bundits och den klyvda produkten frigörs och hur påverkar dessa interaktioner katlysen? Vilken konformation har RNas P i ”transition-state” och hur påverkar denna konformation klyvning-en? Exakt vad för roll har de tvåvärda metalljonerna? Hur mycket bidrar specificitetsdomänen (S) och hur mycket bidrar den katalytiska C-domänen till katalysen och vilken roll har de haft i evolutionen av RNas P? Vilken funktion har RNas P-proteinerna?

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Målsättningen för arbetet som den här avhandlingen baseras på var att förstå hur klyvnigsmekanismen och substratinteraktionen fungerar i RNas P-medierad katalys, med fokus på hur de två RNas P-domänerna (S och C) bidrar till klyvningen, samt vilken betydelse basen i position -1 i substraten (omedelbart innan klyvningsstället i det omogna tRNA:t) har. Jag har huvud-sakligen använt mig av RNA-komponenten hos RNas P i mina studier. Jag har använt mig av biokemiska verktyg, samt olika konstruktioner och mutanter av RNas P-RNA och olika modellsubstrat för att få användbar in-formation om katalysen. Vi har jämfört data från olika RNas P-RNA-substratkombinationer med avseende på t. ex. Mg2+-profiler, val av klyv-ningsställe, och klyvningshastighet. Med hjälp av dessa data utvärderade vi sedan olika tänkbara klyvningsmekanismer för RNas P. De olika RNas P-RNA jag studerat var RNas P-RNA (RPR) från bakterien Escherichia coli (M1 RNA) och från arkéen Pyrococcus furiosus (Pfu), med och utan respek-tive proteiner. Som substrat använde jag mig av olika modellsubstrat basera-de på tRNA-molekyler. Tidigare studier har visat att tRNA redan i omogen form är ihopvikt till sin karakteristiska form och att RNas P känner igen och klyver den (Chang et al. 1973). TSL-regionen (T-stem-loop) i omogna bakterie-tRNA och TBS-regionen (TSL-binding site) i RPR interagerar när RPR-substratkomplexet bildas. Våra data visar att en produktiv TSL/TBS-interaktion påverkar hän-delserna vid klyvningsstället genom att påverka hur de inblandade kemiska grupperna och/eller Mg2+ positioneras så att en effektiv och korrekt sker. Detta överensstämmer med en klyvningsmekanism som kallas ”induced fit” (delarbete I och II). Vidare har våra data visat att den katalytiska domänen (C-domänen) kan klyva substraten i frånvaro av specificitetsdomänen (S-domänen). Klyvning i närvaro av S-domänen, som innehåller TBS-regionen, gav en ökad klyv-ningseffektivitet, men till priset av en försämrad noggrannhet. S-domänen från Pfu RPR interagerar annorlunda med substraten jämfört med S-domänen från M1 RNA. Intressant nog så beter sig en hybrid-RPR där S-domänen från M1 RNA satts ihop med C-domänen från Pfu RPR mest likt M1 RNA. Vi kom fram till att Pfu-proteiner som binder till S-domänen tar över RPR:s roll. Detta är viktigt ur en evolutionär aspekt. Klyvningen påver-kades också när S-domänen var utbytt eller borttagen vilket överensstämmer med ”induced fit”-modellen. Omogna tRNA från Escherichia coli som har en uracil i position -1 är “bätt-re” substrat än omogna tRNA som har andra baser i den positionen. Basen i -1, och då särskilt karbonylsyret i basens position 2 på U-1, bidrog mest till effektiviteten och noggrannheten för klyvningsprocessen, och till att stabili-

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sera transition state. En purin i position -1, speciellt den exocykliska aminen i basens position 2, har en negativ inverkan på klyvningen. En möjlighet är att identiteten av basen i position -1 påverkar laddningsfördelningen och därmed påverkas positioneringen av Mg2+-jonerna vilket resulterar i ett änd-rat klyvningsmönster. Med våra data som bakgrund föreslår vi en konforma-tionsassisterad mekanism som modell för RNas P-medierad klyvning.

54

Acknowledgements

My greatest thanks goes to my advisor, Leif A.K. Without you this work would never have been possible. I have learned a lot from you, both in sci-ence and life. I like the passion that you have in science. I will never forget you said: Life is a bitch then you die. You should kill the bitch and move on.

This is cool! Thank you, Anders V. for all the discussions in science. Thank you, Maria S. Thank you for your great help with my thesis writing and my work! You are my model of the perfect female scientist. I know now that science and family can both co-exist in one’s life. Thank you, people in our group! Ulrika, how can I say thank you ade-quately! You make me feel warm! Mattias B. thank you to being so patient teaching me at the beginning; that was important for me. Ema K., thank you for sharing your knowledge with me. Fredrik P, my personal translator , thank you for all your great help in work and thesis writing! I like all the fun chats! Jaydip G., you taught me a lot! Chen Yu, you are so sweet. Davi-nanders Väg, Mao Guanzhong, Mohammad.F, Fredrik M., Evgen B. thank you for being nice and interesting. Diarmaid H., thank you for teaching me and being so nice. Staffan S. Ger-hart W. Santanu D., thank you for being such knowledgeable PI. Nora A., thank you for all the chats. Suparna S. and Ray, thank you for your great help. Chandra S.M. and Ravi K. K., thank you for your great help. Chen-hui Huang, thank you for your help. Akiko You make me like Japan so much! Eva thanks for making my work so much easier. Nils E. M., thank you so much for teaching me and being so patient, and being a such good person! Urszula thank you for teaching me and the won-derful time! Ulla U. and Hans E, thank you for teaching me and being so nice; the time in GRENOBLE was wonderful! My special thanks goes to Terese B., thank you for all the help with my work and thesis! You are so awesome!

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Thank you, all my friends! All my Adrenalin Junkies! I have been thinking how to write this part since last year. I still cannot completely express my gratitude for having you as my friends! You make me alive, shining, happy, and give me the courage to keep on going in both science and life; you made me! Amanda, pity the Italy trip never happened ! Aurelie, I admire you and I miss all the chats with you! Cia, I wish I were as strong as you (I mean mentally) ; you make me feel safe! Disa, you make me feel like home! Emma, not many people understand me as you do! Hava, my dude, you know what I mean! Marie, my cool idol, how much I wish I could be like you! Nadja, you little sweetie! Yang, my lovely friend! Fabien, Klas and Magnus, how lucky I was to be invited to sit in the same office with you! I enjoyed the fights for the music in the lab, all the jokes you made, all the loud laughs we had, all the late evenings we spent in the lab, all the after work late, outside dinners and all the knowledge you taught me! What a time! Prune, do you really have a tail ? Christina R., you are lovely! Erik and Johan R., thanks for all the wonderful times! Lei and Jia Mi, thank you for all the help and chats. Pernilla, Chris T., thanks for all the nice chats. Henrik T. I like your painting! Hurry, hurry ! Kristin, thank you. Per and Niklas, thank you for all the chats. Rebecka and Kasia, thanks for the nice teaching and wonderful time. Robert F. thanks for being such a lovely PI! The most important thing is to be flexible, right? Lotta, Sonchita, Johan A., Buppi, thank you. Andrea and Fre-drik S, thank you for your help. Patricia, Linda, Helien, Pontus, Adam, Karin T., thank you for the nice time. Petter, Britta, Elin, thank you for being there. Magnus Lundgren, Mikke and Doreen, thank you. Yanhong Pang, Kaweng Ieong, thank you for the nice time. Vasili thank you for ask-ing me how is going. Linda and Mattias, it is nice to have you back to Sweden. Thank you all my friends outside work. You added so much color in my life! My parents.

Zhibing, you are special to me. Thank you for all the support and for being there fore me.

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