1.The Nature of Ribonucleic acid (RNA)- 1 hour

2.Post-transcriptional Regulation- 2 hours

Lectures cover:

Chapter 10

Nucleotides and NucleicAcids

Biochemistry

by

Reginald Garrett and Charles Grisham

• are biological molecules that possess heterocyclic nitrogenous bases

• Nucleotide: see below

• Nucleic acid: linear polymer of nucleotides; serve a central biological purpose – information transfer in cells

1. Deoxyribonucleic acid (DNA)2. Ribonucleic acid (RNA): serve in the expression of genetic

information stored in DNA through the processes of transcription and translation

Nucleotides and Nucleic Acids

• Genetic information is stored in DNA

• Information encoded in a DNA molecule is transcribed via synthesis of an RNA molecule

• The sequence of the RNA molecule is "read" and is translated into the sequence of amino acids in a protein

Importance of DNA and RNA

Figure 10.1 The fundamental process of information transfer in cells. Information encoded in the nucleotide sequence of DNA is transcribed through synthesis of an RNA molecule whose sequence is dictated by the DNA sequence. As the sequence of this RNA is read (as groups of three consecutive nucleotides) by the protein synthesis machinery, it is translated into the sequence of amino acids in a protein. This information transfer system is encapsulated in the dogma: DNA → RNA → protein.

Information Transfer in Cells

What constitute nucleotide (nucleic acid)?

• Complete hydrolysis of nucleotide or nucleic acid liberates:

1. nitrogenous base

2. five-carbon sugar

3. phosphoric acid

in equal amount

• A nucleotide is composed of a nitrogenous base,

a five-carbon sugar, and a phosphate

Nitrogenous base

• Two kinds of nitrogenous base are found in nucleotides

1. pyrimidines: six membered heterocyclic aromatic ring containing two nitrogens

- two pyrimidines are found in RNA: cytosine and uracil

2. purines: 2 rings; one resembles pyrimindine ring

and the other resembles imidazole ring- two purines are found in both RNA and DNA:

adenine and guanine

Figure 10.2 (a) The pyrimidine ring system; by convention, atoms are numbered as indicated. (b) The purine ring system, atoms numbered as shown.

Figure 10.3 The common pyrimidine bases—cytosine, uracil, and thymine—in the tautomeric forms predominant at pH 7.

Figure 10.4 The common purine bases—adenine and guanine—in the tautomeric forms predominant at pH 7.

The Sugar (Ribose)

• The sugar is pentose (five-carbon sugar)

• Ribonuceloside- D-ribose → five-membered ring known as furanose (D-ribofuranose)

• Deoxyribonucleoside- 2-deoxy-D-ribose → five-membered ring (2-deoxy-D-ribofuranose)

• Atoms are numbered as 1’, 2’, 3’

Figure 10.9  Furanose structures—ribose and deoxyribose.

• A nucleoside = a nitrogenous base + a sugar

• Base is linked to sugar via a glycosidic bond

• Named by adding -osine to the root name of a purine and -idine to the root name of a pyrimidine

What Is Nucleoside?

Bases Nucleosides

adenine adenosineguanine + Sugar guanosine

cytosine cytidineuracil uridine

Figure 10.10 b-Glycosidic bonds link nitrogenous bases and sugars to form nucleosides.

Figure 10.11 The common ribonucleosides—cytidine, uridine, adenosine, and guanosine. Also, inosine drawn in anti conformation.

What Is Nucleotide?

• Nucleoside (base+sugar) + phosphate group

• Phosphoric acid is esterified to a sugar’s OH group

• C-5’ OH is esterified with phosphoric acid in the cell; a ribonucleotide has a 5’ phosphate group

• The number of phosphate at the C-5’ can be 1, 2, or 3

Figure 10.13 Structures of the four common ribonucleotides—AMP, GMP, CMP, and UMP—together with their two sets of full names, for example, adenosine 5'-monophosphate and adenylic acid. Also shown is the nucleoside 3'-AMP.

Figure 10.15 Formation of ADP and ATP by the successive addition of phosphate groups via phosphoric anhydride linkages. Note the removal of equivalents of H2O in these dehydration synthesis reactions.

Base Nucleoside Nucleotide

adenine adenosine adenosine 5’-monophosphate (AMP)adenosine 5’-diphosphate (ADP)adenosine 5’-triphosphate (ATP)

guanine guanosine GMP, GDP, GTP

cytosine cytidine CMP, CDP, CTP

uracial uridine UMP, UDP, UTP

What Is Nucleic Acid?

• Polymer of nucleotides linked 3’ to 5’ by phosphodiester bridges – formed as nucleoside 5’- monophosphate is successively added to the 3’ OH group of the preceding nucleotide

• Polymer of ribonucleotides = RNA

• The convention way to read and write the polynucleotide chain is from the 5’ end to the 3’ end

• Note that this reading actually passes through each phosphodiester from 3’ to 5’

Figure 10.173'-5' phosphodiester bridges link nucleotides together to form polynucleotide chains.

Figure 10.18Furanoses are represented by vertical lines; phosphodiesters are represented by diagonal slashes in this shorthand notation for nucleic acid structures.

TGCAT

What Are the Different Classes of Nucleic Acids?

• DNA - one type, one purpose • RNA - 3 (or 4) types, 3 (or 4) purposes

– messenger RNA (mRNA) – ribosomal RNA (rRNA)– transfer RNA (tRNA)– Small nuclear RNA (snRNA) - pre-mRNA splicing– Small non-coding RNAs - post-transcriptional

gene silencing

Table 10-02, p.319

Messenger RNA (mRNA)

• Serves to carry information that is encoded (stored) in DNA to the sites of protein synthesis

• In prokaryotes, a single mRNA contains the information for synthesis of many proteins

• In eukaryotes, a single mRNA codes for just one protein, but structure is composed of introns and exons

Eukaryotic mRNA

• DNA is transcribed to produce heterogeneous nuclear RNA – mixed introns and exons with poly A– intron - intervening sequence – exon - coding sequence– poly A tail - stability?

• Splicing produces final mRNA without introns

Ribosomal RNA (rRNA)

• Ribosomes are about 2/3 RNA, 1/3 protein

• rRNA serves as a scaffold for ribosomal proteins

• Form complex secondary structures

• Their relative sizes are referred to as sedimentation coefficients (S)

p.323

Figure 10.25 The organization and composition of prokaryotic and eukaryotic ribosomes.

Transfer RNA tRNA)

• Small polynucleotide chains - 73 to 94 residues each • Several bases usually methylated• Fold into a characteristic secondary structure• Each a.a. has at least one unique tRNA which carries

the a.a. to the ribosome • 3'-terminal sequence is always CCA-a.a. • Aminoacyl tRNA molecules are the substrates of

protein synthesis

p.325

DNA & RNA Differences?

Why is DNA 2'-deoxy and RNA is not?

• Vicinal -OH groups (2' and 3') in RNA make it more susceptible to hydrolysis

• DNA, lacking 2'-OH is more stable

• This makes sense - the genetic material (DNA) must be more stable

• RNA is designed to be used and then broken down

Hydrolysis of Nucleic Acid

• RNA is resistant to dilute acid

• RNA is hydrolyzed by dilute base

• DNA and RNA are hydrolyzed by nucleases

Figure 10.29The vicinal -OH groups of RNA are susceptible to nucleophilic attack leading to hydrolysis of the phosphodiester bond and fracture of the polynucleotide chain; DNA lacks a 2'-OH vicinal to its 3'-O-phosphodiester backbone. Alkaline hydrolysis of RNA results in the formation of a mixture of 2'- and 3'-nucleoside monophosphates.

Nucleases

• Enzymes that hydrolyze nucleic acids - DNase and RNase

• Nucleases are phosphodiesterase that cleave phosphodiester bonds by using H2O

• Cleavage can be occurred on either side of the phosphorus; at the 3’ side is labeled as “a” and at the 5’-side labeled as “b”

• Cleave internally is called endo; cleave from terminal nucleotides is called exo

• Exo a: cleave at “a” from the 3’ end - snake venom phosphodiesterase

• Exo b: cleave at “b” from the 5’ end – spleen phosphodiesterase

Figure 10.30 Cleavage in polynucleotide chains: a cleavage yields 5'-phosphate products, whereas b cleavage gives 3'-phosphate products.

Figure 10.31 Snake venom phosphodiesterase and spleen phosphodiesterase are exonucleases that degrade polynucleotides from opposite ends.

Figure 10.32 An example of nuclease specificity: The specificity of RNA hydrolysis by bovine pancreatic RNase. This RNase cleaves b at 3'-pyrimidines, yielding oligonucleotides with pyrimidine 3'-PO4 ends.

• Pancreatic RNase A: an endo ribonuclease cleaves “b” after pyrimidines (C or U)

Post-Transcriptional Regulation

• Biochemistry – Fourth Edition by Garrett & Grisham

• Chapter 29 – Pages 974-981

Figure 29.1Crick’s 1958 view of the “central dogma of molecular biology”: Directional flow of detailed sequence information includes DNA®DNA (replication), DNA®RNA (transcription), RNA®protein (translation), RNA®DNA (reverse transcription). Note that no pathway exists for the flow of information from proteins to nucleic acids, that is, proteins®RNA or DNA. A possible path from DNA to protein has since been discounted. Interestingly, in 1958, mRNA had not yet been discovered.

rRNAs constitute ~80-90% of total cellular RNAs

mRNAs only constitute ~3-5% of total cellular RNAs

What is post-transcriptional regulation ?

• Transcription: The process of RNA synthesis.

• Post-transcriptional regulation: The regulation AFTER transcription, but not translation.

• AFTER DOES NOT mean that transcription has to be complete.

How Are Eukaryotic Transcripts Processed and Delivered to the Ribosomes for Translation?

• In prokaryotes, transcription and translation are concomitant processes

• In eukaryotes, the two processes are spatially separated: transcription occurs on DNA in the nucleus, and translation occurs on ribosomes in the cytoplasm

• Thus, transcripts must be transported from the nucleus to the cytosol to be translated

• On the way, these transcripts undergo processing– Alterations that convert the newly synthesized RNAs

(primary transcripts) into mature mRNAs• And unlike prokaryotes, eukaryotic mRNAs encode only one

polypeptide; i.e., they are monocistronic

mRNA degradation mRNA localization

post-transcriptional regulation

Structural and spatial difference in gene expression between procaryotes and eucaryotes

Comparison between procaryotic and eucaryotic mRNAs

polycistronic

monocistronic

• Introns intervene between exons

• Exon: protein coding region; intron: noncoding region

• Exon size is much smaller than intron size

• Examples: actin gene has 309-bp intron separates first three amino acids and the other 350 or so

• Chicken pro-alpha-2 collagen gene is 40-kbp (40,000 bp) long, with 51 exons of only 5 kbp total; the exons range in size from 45 to 249 bases

• Most introns (lengths vary from 60-10,000 bps) are untranslatable; they need to be removed – splicing

• Mechanism by which introns are excised and exons are spliced together is complex and must be precise

Eukaryotic Genes are Split Genes

Eukaryotic Genes are Split Genes

Figure 29.36 The organization of split eukaryotic genes.

The organization of the mammalian dihydrofolate reductase (DHFR) gene

The gene is split into 6 exons spread over 31-kbpThe six exons are spliced together to give a 6-kb mRNANote that the size of the exons are much shorter than the introns, and the exon pattern is more highly conserved than the intron pattern

Nuclear mRNA Processing Involves:

1. Capping and Methylation 2. Polyadenylylation3. Splicing

Post-transcriptional regulation in the nucleus

• Primary transcripts (pre-mRNAs) are "capped“ as soon as they are transcribed by RNA polymerase II

• The reaction is catalyzed by guanylyl transferase using GTP as a substrate

• Capped G residue is methylated at N7-position

• Additional methylation occurs at C2'-O positions of next two residues and at 6-amino group of the first adenine (if A is the initial nucleotide)

Capping and Methylation

Figure 29.37The capping of eukaryotic pre-mRNAs. Guanylyl transferase catalyzes the addition of a guanylyl residue (Gp) derived from GTP to the 5¢-end of the growing transcript, which has a 5¢-triphosphate group already there. In the process, pyrophosphate (pp) is liberated from GTP and the terminal phosphate (p) is removed from the transcript. Gppp + pppApNpNpNp… ® GpppApNpNpNp… + pp + p (A is often the initial nucleotide in the primary transcript).

The Capping of Eukaryotic pre-mRNAs

Figure 29.38Methylation of several specific sites located at the 5¢-end of eukaryotic pre-mRNAs is an essential step in mRNA maturation. A cap bearing only a single ¾CH3 on the guanyl is termed cap 0. This methylation occurs in all eukaryotic mRNAs. If a methyl is also added to the 2¢-O position of the first nucleoside after the cap, a cap 1 structure is generated. This is the predominant cap form in all multicellular eukaryotes. Some species add a third ¾CH3 to the 2¢-O position of the second nucleoside after the cap, giving a cap 2 structure. Also, if the first base after the cap is an adenine, it may be methylated on its 6-NH2. In addition, approximately 0.1% of the adenine bases throughout the mRNA of higher eukaryotes carry methylation on their 6-NH2 groups.

Cap 0

Cap 1

Cap 2

Phosphatase

Guanylyl transferase

Guanine 7-Methyl transferase

2’ O-Methyl transferase

Enzymes involved in the Capping

Why do cells need to cap their mRNA?

- cap is recognized by cap-binding proteins

- cap distinguishes mRNAs from other types of RNA molecules (RNA pol I and III produce uncapped RNAs)

- mRNAs need a cap (and poly A tail) for export from the nucleus

- cap is necessary for translation

- cap stabilizes mRNA in the cytoplasm

3'-Polyadenylation and transcriptional termination

• Termination of transcription occurs only after RNA polymerase has transcribed a consensus AAUAAA

sequence - the poly(A) signal

• 10-30 nucleotides after this site [the poly(A) signal], the mRNA is cleaved and a string of ~200 adenine residues is added to the mRNA transcript - the poly(A) tail

• poly(A) polymerase adds these A residues

• poly(A) tail bound by PABP stimulates translation and

governs the stability of mRNA

3'-Polyadenylation

Signals required for the formation of the 3’ end of mRNA

Figure 29.39Poly(A) addition to the 3¢-ends of transcripts occurs 10 to 35 nucleotides downstream from a consensus AAUAAA sequence, defined as the polyadenylylation signal. CPSF (cleavage and polyadenylylation specificity factor) binds to this signal sequence and mediates looping of the 3¢-end of the transcript through interactions with a G/U-rich sequence even further downstream. Cleavage factors (CFs) then bind and bring about the endonucleolytic cleavage of the transcript to create a new 3¢-end 10 to 35 nucleotides downstream from the polyadenylylation signal. Poly(A) polymerase (PAP) then successively adds 200 to 250 adenylyl residues to the new 3¢-end. (RNA polymerase II is also a significant part of the polyadenylylation complex at the 3¢-end of the transcript, but for simplicity in illustration, its presence is not shown in the lower part of the figure.)

Polyadenylation of mRNA

A. Where is the template? - does not require a template - the poly(A) tail is not encoded in the genome

B. What’s the function of the polyA tail? - by interaction with poly(A) binding protein (PABP),

it is necessary for efficient translation and protection from mRNA degradation

Pre-mRNA splicing

splicing

translation

Nuclear Pre-mRNA Splicing

• Within the nucleus, pre-mRNA forms ribonucleoprotein particles (RNPs) through association with a characteristic set of nuclear proteins

• These proteins maintain the pre-mRNA in an untangled and accessible conformation

• The substrate for splicing, that is, intron excision and exon ligation, is the capped primary transcript emerging from the RNA polymerase II transcriptional apparatus

• Splicing occurs exclusively in the nucleus

• Consensus sequences define the exon/intron junctions in eukaryotic mRNA precursors

Splicing of Pre-mRNA

Capped, polyadenylated RNA, in the form of a RNP complex, is the substrate for splicing

• In "splicing", the introns are excised and the exons are sewn together to form mature mRNA

• The 5'-end of an intron in higher eukaryotes is always GU and the 3'-end is always AG

• All introns have a "branch site" 18 to 40 nucleotides upstream from 3'-splice site

• The branch site is essential to splicing

5’ splice site

3’ splice site

Branch site (usually closer to 3’ss)

What makes an intron?

R: Purine A or GY: Pyrimidine U or C

The Splicing Reaction Proceeds via Formation of a Lariat Intermediate

• Next slide shows the splicing mechanism

• The branch site is usually YNYRAY, where Y = pyrimidine, R = purine and N is anything

• The lariat a covalently closed loop of RNA is formed by attachment of the 5'-P of the intron's invariant 5'-G to the 2'-OH at the branch A site

• The exons then join, excising the lariat

• The lariat is unstable; the 2'-5' phosphodiester is quickly cleaved and the intron is degraded in the nucleus

The Splicing Reaction Proceeds via Formation of a Lariat Intermediate

Degraded

Splicing Depends on snRNPs

• Splicing depends on a unique set of small nuclear ribonucleoprotein particles - snRNPs, pronounced "snurps"

• A snRNP consists of a small RNA (100-200 bases long) and about 10 different proteins

• Some of the 10 proteins are general, some are specific (see Table 29.6)

• Major snRNP species are abundant, with more than 100,000 copies per nucleus

• snRNPs and pre-mRNA form the spliceosome

Splicing Depends on snRNPs

snRNPs Form the Spliceosome

• Splicing occurs when the various snRNPs come together with the pre-mRNA to form a multicomponent complex called the spliceosome

• The spliceosome is a large complex, about the size of a ribosome; its assembly requires ATP

• snRNP U1 binds at the 5'-splice site, and U2 snRNP binds at the branch site

• Interaction between the snRNPs brings 5'- and 3'- splice sites together so lariat can form and exon ligation can occur

• The transesterification reactions that join the exons may in fact be catalyzed by snRNAs themselves, but not by snRNP proteins

• Spliceosome assembly requires ATP-dependent RNA rearrangements catalyzed by spliceosomal DEAD-box ATPases/helicases

The spliceosome - RNA/protein complex

Composition:

Small nuclear RNAs (snRNAs):

5 snRNAs (U1, U2, U4, U5, U6), uridine rich, range in size from 106-189 nucleotide long.

Proteins:

10 identified associated with each of the snRNAs to form the small nuclear ribonucleoproteins (snRNPs)

non-snRNP splicing factors: SR-family proteins and other splicing factors

(current estimate of total proteins is >50 different)

snRNPs Form the Spliceosome1st event: U1 interacts with the 5’ splice site

Figure 29.43 Mammalian U1 snRNA can be arranged in a secondary structure where its 5'-end is single-stranded and can base-pair with the consensus 5'-splice site of the intron.

Figure 29.44 Events in spliceosome assembly. U1 snRNP binds at the 5'-splice site, followed by the association of U2 snRNP with the UACUAA*C branch-point sequence. The triple U4/U6-U5 snRNP complex replaces U1 at the 5'-splice site and directs the juxtaposition of the branch-point sequence with the 5'-splice site, whereupon U4 snRNP is released.

Assembly of the spliceosome

Alternative Splicing Creates Protein Isoforms

• In constitutive splicing, every intron is removed and every exon is incorporated into the mature RNA

• This produces a single form of mature mRNA from the primary transcript

• However, many eukaryotic genes can give rise to multiple forms of mature RNA transcripts

• This may occur by:– Use of different promoters– Selection of different polyadenylylation sites– Alternative splicing of the primary transcript, or– A combination of these three mechanisms

Constitutive Splicing vs. Alternative Splicing

Constitutive mRNA Splicing

Every intron is removed and every exon is incorporated into mature RNA without exception – results in a single form of mature mRNA

I II III

I II III

A cell

I II III

I II III

B cell

Alternative mRNA splicing I

Both isoforms are expressed in both A and B cell

I II III

I II III

A cell

I II III

I II III

B cell

I III I III

Alternative mRNA splicing II

Different cell types express distinct isoform

I II III

I II III

A cell

I II III

I II III

B cell

I III

I II III

C cell

I III

Alternative mRNA Splicing Creates Protein Isoforms

• Different transcript from a single gene make possible a set of related polypeptides, termed protein isoforms, each with a slightly altered function

• The isoforms of fast skeletal muscle troponin T are an example of alternative splicing

• This gene consists of 18 exons, 11 of which are found in all mature mRNAs and are constitutive

• Five of the exons (4 through 8) are combinatorial, in that they may be included or excluded

• Two (16 and 17) are mutually exclusive – one is always present but never both• 64 different mature mRNA can be formed from this gene by alternative splicing

Alternative splicing expands the coding potential of the genome – Tissue-specific splicing

7mGppp AAAAAA

mRNA export

mRNA processing

mRNA Export

5’ end of mRNA leaves the nucleus first. Note exchange of some of the proteins which do not leave the nucleus.

Fates of mRNAs in the cytoplasm

• Translation

• Degradation

• Localization → translation

Post-transcriptional regulation in the cytoplasm

The competition between mRNA translation and mRNA decay

- The mRNA is thought to be circularized by its interaction with eIF4E, eIF4G, and PABP- This interaction stimulates mRNA translation and protects the 5’ and 3’ ends of the mRNA from attack by decay enzymes- Any factors affecting translation efficiency will have an opposite effect on its degradation

Poly(A) nuclease: removes poly(A) tail

Two mechanisms of eucaryotic mRNA decay

General decay pathway: most mRNAs Certain mRNAs: requires specific sequences

mRNA localization

Generally mRNAs are translated in the cytoplasm by free ribosomes; their products may be directed to other sites in the cell

mRNAs encoding secreted or membrane-bound proteins are directed to endoplasmic reticulum (ER) by a signal at the amino terminus of the protein

Some mRNAs are directed to specific intracellular locations before translation begins, i.e. translated at the site where the protein functions

Basic features of mRNA localization include cis-acting elements within the mRNA that targets the message to a subcellular region, a protein-RNA complex that effects localization, and the cytoskeleton that acts as a “road” for RNA movement

Most localization signal sequences appear to be in the 3'UTR (untranslated region) of mRNAs

The importance of 3’ UTR in mRNA localization

mRNA encoding hairy protein are normally localized to the apical site of nuclei

Injected hairy RNA containing the 3’ UTR

Injected hairy RNA lacking the 3’ UTR

5’UTR 3’UTR Coding Region

5’UTR Coding Region

RNA Editing: Another Way To Increase the Diversity of Genetic Information

• RNA editing is a process that changes one or more nucleotides in an RNA transcript by deaminating a base, either A→I or C→U

• These changes alter the coding possibilities in a transcript, because I will pair with G (not U as A does) and U will pair with A (not G as C does)

• RNA editing can increase protein diversity by

(1) altering amino acid coding possibilities

(2) introducing a premature stop codon

(3) changing splice site in a transcript

RNA editing – A to I editing

Carried out by ADAR- adenosine deaminase acting on RNA, which recognizes a double-stranded RNA region

Such regions form when an exon region containing the A to be edited base pairs with a complementary base sequences in an intron known as the editing site complementarysequence

A U

I C

---

------

---

Editing of apolipoprotein B (apoB) mRNA – C to U

Non-edited mRNA encodes 550-kDa protein; functions to make liver-derived VLDL

Editing of CAA codon to UAA stop site at codon 2152; edited mRNA encodes 250-kDa protein; functions to make intestinal-derived lipid complexes

Carried out on a single-stranded regions oftranscript by an editosome whose core structure consists of a cytosine deaminase and an adapter protein that brings the deaminase and the transcript together

Stages: transcription → transcript processing → mRNA export → translation

Traditionally they have been presented as a linear series of events (a pathway of discrete and independent steps) – each going to completionbefore the next begins

Now it is clear that each stage is part of a continuous process with physical and functional connections between the transcriptional and processing machineries.

Capping, RNA splicing, 3’ end formation and polyadenylation, and nuclear export are coupled to transcriptional machinery

Regulation occurs at multiple levels in this continuous processin a coordinated fashion

Eucaryotic cells have elaborate mRNA surveillance systems to destroy any messages containing errors

Unified theory of gene expression