Principles of Microbial Molecular Biologypeople.uleth.ca/~selibl/Biol3200/CourseNotes/MicroGenCh8... · Web viewProkaryotic DNA fewer proteins associated with it than eukaryotic DNA

Biol 3400Tortora et al Chap 8

Microbial Genetics

Genetics - the study of the mechanisms by which traits are passed from one organism to another and how they are expressed

Why study the flow of biological information? Understanding the molecular mechanisms by which cells work, species diversity and

evolution Production of genetically modified organisms (GMO – research and practical applications) Medical diagnosis

Information flow in the cell (Figure 8.2) One way flow of information Flow of information from one generation to the next (DNA replication) Gene expression = DNA RNA Protein (central dogma of molecular biology)

I. DNA StructureReview – Chapter 2, Figure 2.16

Right handed double helix Complementary antiparallel polymers (strands) of nucleotides Can form other secondary structures such as hairpins and stem-loop structures

Prokaryotic DNA fewer proteins associated with it than eukaryotic DNA supercoiling is the result of DNA gyrases (topisomerases) recall targets for quinolones (e.g., nalidixic acid, ciprofloxacin) and novobiocin DNA gyrase (topoisomerase II) introduces the negative supercoiling Topisomerase I uncoils the supercoiling

Eukaryotic DNA Tightly wound around histone (positively charged proteins) complexes – 166 bp in 1¾ turns Core histones are composed of eight polypeptides (2 copies of histones H2A, H2B, H3 and

H4). Histone/DNA complexes form regularly spaced nucleosomes thereby introducing negative

supercoiling. Histone H1 attaches to the DNA near the nucleosome. There is further folding and looping associated with a protein scaffold.

Archaeal DNA DNA is complexed with archaeal histones to form nucleoprotein complexes called archaeal

nucleosomes (4 histone-like proteins)

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II. DNA Replication Semi-conservative process for the synthesis of DNA In general, DNA replication in Archaea is more similar to eukaryotic cells than bacterial cells Recall cellular differences: eukaryote vs prokaryote Review (Figures 8.3 to 8.6; Table 8.1)

Replication rates 750-1000 b/s for bacteria and 50 to 100 b/s for eukaryotes

1. Major components fo DNA replication DNA template RNA primers Enzymes and other proteins

DNA polymerases – (III & I in E. coli) – DNA Pol III holoenzyme is composed of 10 proteins with the core being composed of , , subunits

Helicase Single-strand binding protein Primase DNA ligase Topoisomerases - unwinding and supercoiling the DNA molecules – important in

regulating replication

2. Steps involved in DNA replication (Fig 8.5)

i) Initiation of DNA synthesis initiates at the origin of replication by specific initiation proteins - one origin on a circular

prokaryotic chromosome (oriC ~300 bp sequence in E. coli) to many (every 30 to 300 kb) on a linear eukaryotic chromosome. Note: several species of Sulfolobus have more than one origin.

DnaA proteins bind to specific nucleotide sequences (DnaA boxes) within the origin and hydrolyze ATP to break the hydrogen bonds; Helicase opens up the DNA strands to form a replication bubble; Topoisomerases (i.e., DNA gyrase in bacteria) relieve the tension generated by unwinding the DNA; single-stranded DNA binding protein stabilize the open structure;

RNA primer (usually about 10 nt) added by a primase (requires several other proteins that are found in the primosome)

ii) Elongation (5' 3') DNA polymerase (DNA Pol III in E. coli) adds nucleotides to the 3'-OH end of RNA primer

or growing DNA polymer. Error rates in DNA synthesis are low (10-8 to 10-11) due to the proofreading that is undertaken

by DNA polymerase III as it synthesizes the new DNA strand. 3'5' exonuclease activity removes incorrect nucleotide.

Leading and lagging strand synthesis occur within a duplex of DNA polymerases (replisome consisting of helicases, primosome, two DNA polymerase III molecules and other associated proteins) – The DNA strands are pulled through this protein complex

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Okazaki fragments are about 1000 to 2000 nt in bacteria and 100 nt in eukaryotesiii) Joining or Termination DNA polymerase I, using 5’ to 3’ exonuclease activity, replaces RNA primers with DNA DNA ligase forms phosphodiester bond between 3’ –OH and 5’ -PO4 of adjacent DNA

molecules

Replication stops when the replisome reaches the termination site (ter)

In organisms with linear DNA molecules, these linear How is the 5' end of linear molecules replicated? Circularization of the linear molecule viruses such as lambda Protein primers some DNA polymerases can add nucleotides onto 3' -OH found on

specific proteins (viruses and linear plasmids) Eukaryotes telomeres contain repetitive DNA (often 6 bp repeats). Telomerase adds on

these repeated sequences onto the 3' end of the linear DNA thereby extending the strand so that there is enough DNA to allow for the addition of an RNA primer.

III. Gene Expression

What is a gene – a polynucleotide sequence that codes for a functional product (i.e., a polypeptide, tRNA or rRNA)

The basic components of a bacterial gene include the promoter, leader, coding regions and termination region including trailer and terminator sequences as well as a number of possible regulatory sites

Many eukaryotic genes and a few archaeal genes contain coding regions (exons) that are periodically interrupted by noncoding sequences (introns). The introns are cut out of the transcript before the polypeptide is made. Alternative splicing is a process that involves selective splicing of introns thereby facilitating the production of more than one polypeptide from a single gene.

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A. Transcription

DNA RNA (Figure 8.7) Only one strand of DNA is transcribed - template or antisense strand. The other strand is

called the coding or sense strand, i.e., it has the same sequence as the mRNA except in DNA bases.

(DNA dependent) RNA polymerases use the template strand to synthesize the mRNA in a 5' 3' direction - form phosphodiester bonds between two ribonucleotides. No primer is required. First base is almost always a purine (A of G)

Takes place in the nucleus in eukaryotic cells and in the cytoplasm in prokaryotic cells

Bacteria and Archaea have one type of RNA polymerase - synthesizes all types of RNA including mRNA

E. coli core enzyme - 5 core polypeptides or subunits - 2 , , ' and sensitive rifampicin and streptolydigin - binds to subunit factor involved in initiation of RNA synthesis; required for specificity and recognition of

transcriptional start site (e.g., there is a sigma factor cascade involved in regulating sporulation)

(omega) factor of unknown function core + RNA polymerase holoenzyme: capable of initiating transcription

Archaea contain only one RNA polymerase that is unique to these microbes more closely related to eukaryotes 8 to 12 polypeptides insensitive to rifampicin and streptolydigin

Eukaryotes produce three types of RNA polymerases (I, II, and III) complex - composed of 9 - 12 subunits requires extra transcription factors to recognize promoters

RNA polymerase I - rRNARNA polymerase II - mRNARNA polymerase III - tRNA and 5.8S rRNA

sensitive to amanitin but insensitive to rifampicin and streptolydigin

Specific DNA sequences mark initiation and termination sites for this process DNA sequence that is transcribed is called a transcriptional unit Some transcriptional units may contain more than one gene rRNA genes in both eukaryotes and prokaryotes are found in clusters and all the genes in the

cluster are cotranscribed

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In prokaryotes (i.e., Bacteria and Archaea) a single mRNA molecule may encode more than one polypeptide – polycistronic mRNA. This phenomenon generally does not occur in eukaryotes

1. Steps in Transcription (Fig 8.7)

i) RNA polymerase binding and intiation of transcription RNA polymerase binds to DNA regions called promoters (~100 to 200 bp) promoter regions contain the transcriptional initiation site as well as recognition sites. It

also identifies sense strand. The factor plays a very important role in this process. The RNA polymerase core enzyme

cannot bind tightly or specifically to DNA. The sigma factor facilitates binding of the NA polymerase holoenzyme

Recognition Sequences Bacteria recognition sequences - promoter consensus sequences (conserved sequences)

around the -10 region (Pribnow box - TATAAT) from the transcriptional start site (+1) and the -35 region (TTGACA) with an unconserved spacer between these sequences

Eukaryotic promoter elements - TATA box at about -30 or greater upstream. This region binds additional transcription factors required by RNA polymerase II. These factors are independent of the RNA polymerase. TFIID transcription factor contains the TATA binding protein (TBP) that bends the DNA and makes it more accessible to other initiation factors. Additional promoter elements include GC and CAAT boxes that are located between 50 and 100 bp upstream of the transcription initiation site.

We don’t have a clear understanding of the variety of general transcription factors, promoter specific factors and promoter elements that have been discovered in different eukaryotic cells

Archaeal cells RNA polymerases recognize promoter sequences similar to eukaryotic cells (i.e., TATA box) and require a TATA binding protein. Archaeal RNA polymerase also require several additional transcription factors .

Promoters with nucleotide sequences that favour efficient transcription - strong promoters. The converse is a weak promoter.

Promoter strength depends upon nucleotide sequence and ability of DNA strands to separate

Once the RNA polymerase is bound to the promoter it begins to separate the DNA strands at the initiation site. An unwound region of two turns of the helix (16 – 20 bp) becomes the transcription bubble.

ii) Elongation of the RNA strand The polymerase untwists one turn of the double helix at a time, separates strands and adds

nucleotides to the 3' end of the growing RNA molecule The factor dissociates soon after elongation begins synthesis rate = 40 nt/s compared to DNA synthesis (~ 1000 nt/s) a single gene can be simultaneously transcribed by more than one RNA polymerase

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iii) Termination of transciption transcription continues until the RNA polymerase reaches a termination sequence In bacteria these sequences often contain inverted repeats - stem loop structure will form in

the RNA molecule Proteins may also be involved in termination (e.g., Rho poly-U regions too weak to hold

mRNA:DNA duplex together) AATAAA - common sequence near sites of transcription termination in eukaryotic systems

2. Posttranscriptional modification of RNAs tRNA and rRNA of archaea, bacteria, eukaryotes - originally transcribed molecules are often

much larger mRNA often modified in eukaryotes and rarely in bacteria and archaea mRNA modification (eukaryotes) - removal of introns (intervening sequences) by small

nuclear ribonucleoproteins (snRNPs) and splicing back together the remaining exons. A few Archaeal genes have been shown to have introns.

RNA may act as a catalyst in this process - ribozyme – most are self-splicing introns (vestiges of “RNA life”?)

mRNA from eukaryotes are further modified to include a 5' methylated guanosine triphosphate cap and a 3'-OH poly-A tail.

B. Translation mRNA carries a series of codons which are interpreted during translation to synthesize

polypeptides (a rapid process - 900 or 100 residues/min in E. coli or eukaryotes, respectively) Protein coding sequence is defined by the start (AUG) and a downstream stop (nonsense)

codon (UAA, UGA, UAG) in the same reading frame Open reading frame (ORF) – potential coding sequence starting with a start codon, followed

by a number of codons (in the same reading frame as the start codon) and terminating with the first in frame stop codon.

The genetic code is nearly universal

Components involved in translation

1. Messenger RNA (mRNA) In bacteria the beginning and end of these molecules are often not translated. The beginning sequence or leader sequence is noncoding and involved in the initiation of

protein synthesis; contains the Shine-Dalgarno sequence that is important in initiation of translation. The leader sequence may also be important in regulation of transcription and translation.

In bacterial and archaeal cells transcription and translation can occur simultaneously

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2. Transfer RNA (tRNA) Amino acids are brought to ribosome attached to a transfer RNA molecule (tRNA). In eukaryotes the tRNA are synthesized in the nucleus using DNA as a template and

translocated to the cytoplasm where used repeatedly to shuttle amino acids to the sites of protein synthesis.

Approximately 80 nt long (73-93 nt) folds into a three dimensional L-shape through hydrogen bonds

Anticodon at one end and at the other end the 3'end of the strand protrudes and functions as the site for amino acid attachment

Aminoacyl-tRNA synthetase attaches (charging) amino acids to correct tRNA molecules at the acceptor end (CCA). There is a family of aminoacyl-tRNA synthetases: one for each amino acid. This process requires ATP

The tRNA interprets codons and aligns with the mRNA strand according to a base triplet at one end called the anticodon (complementary to codon on mRNA strand). These molecules carry specific amino acids depending on their anticodon - not all tRNA are identical

3. Ribosomes facilitate the coupling of specific tRNA anticodons and mRNA codons two subunits - a large and a small subunit In eukaryotes, the subunits are assembled in the nucleolus and exported by the nuclear pores The subunits join to form a functional ribosome composition - numerous proteins and ribosomal RNA (60% of the ribosome weight) Prokaryotic and eukaryotic ribosomes are similar in structure and function but differ in size

(Prokaryotic ribosomes are smaller) and composition (antibiotics specific to prokaryotic ribosomes such as tetracycline and streptomycin have no effect on eukaryotic organisms)

Structureo P site (peptidyl-tRNA binding site) holds the growing polypeptide chaino A site (aminoacyl-tRNA binding site) holds the tRNA carrying the next amino acid to be

added to the chain.o E-site (Exit site) site where tRNA are released from ribosomeo Holds the tRNA and mRNA molecules together and catalyzes the addition of an amino

acid to the carboxyl end of the growing polypeptide chain

4. Steps involved in translation (Fig 8.9)

i) Chain initiation mRNA, tRNA carrying the first amino acid of the polypeptide and the two subunits of the

ribosome are brought together

initiation complex - small subunit of the ribosome binds both the mRNA (RBS - Shine-Dalgarno site in bacterial and archaea - AGGAGG - complementary to 3'-OH end of 16S rRNA) and a special initiator tRNA (carries N-formylmethionine in bacteria) attaches to initiation codon (AUG). The Shine-Dalgarno sequence allows prokaryotic ribosomes to translate polycistronic mRNA.

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large ribosomal subunit attaches resulting in a functional ribosome. This step is catalyzed by initiation factors (three initiation factors in bacteria: IF-1, IF-2 and IF-3 and more are required in eukaryotes). Requires energy - 1 GTP.

This step is over when the initiator tRNA sits in the P site with a vacant A site.

Initiation is very elaborate to ensure translation does not begin in the middle of a gene.

ii) Elongation amino acids are added one by one to the initial amino acid. three step cycle requiring several proteins called elongation factors. (60 ms/cycle)

a. Codon recognition - mRNA codon in the A site forms hydrogen bonds with anticodon of an incoming tRNA carrying the appropriate amino acid. Elongation factor EF-Tu moves tRNA into the A site - requires hydrolysis of phosphate bond from GTP

b. Peptide bond formation – 23S rRNA from large ribosomal subunit appears to catalyze the formation of a peptide bond between the polypeptide extending from the P site and the newly arrived amino acid in the A site. Polypeptide separates from the tRNA to which it was bound in the P site and is transferred to the amino acid carried by the tRNA in the A site.

c. Translocation - The tRNA in the P site moves to the E-site and dissociates from the ribosome and the A site tRNA (and carrying the polypeptide) is translocated to the P site. The tRNA remains attached to the mRNA codon. Requires one GTP molecule. The movement places the next codon to be translated in the A site. mRNA is moved through the ribosome in a 5' to 3' direction. EF-G (translocase) is required in this process.

iii. Termination Elongation continues until a stop codon (UAG, UAA, UGA) reaches the A site. Three release factor proteins (RF-1, RF-2 and RF-3) aid the ribosome in recognizing the stop

codons. A release factor protein binds to the stop codon and causes the ribosome to add a water molecule instead of an amino acid to the polypeptide. This frees the polypeptide from the tRNA in the P site.

The ribosome separates into the two subunits. IF-1 binds to the 30S subunit and prevents reassociation to the 50S subunit until the proper stage of initiation is reached.

Polyribosomes - several ribosomes may simultaneously translate the same mRNA.

Prokaryotes may couple transcription and translation

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5. Posttranslational modification Enzymatic changes that modify protein after translation is complete.

Removal of N terminus Met in archaea and eukaryotes Removal of C terminus Modification of individual amino acid residues

Phosphorylation of S, Y and W Glycosylation - occurs frequently in eukaryotes and infrequently in prokaryotes Splicing out amino acids (inteins) leaving exteins.

6. Folding and Secretion Some proteins may require other proteins to fold correctly – molecular chaperones Proteins that are transported through membranes often are synthesized with an extra 15 – 20

amino acids at the N-terminus – signal sequence Signal sequences – few positive residues at the start, followed by central region of

hydrophobic residues and then more polar residues Signal sequence is used by secretory systems to identify proteins that need to be moved

through a membrane. The signal sequence is removed from the proprotein during the secretion process to produce a mature protein

IV. Regulation of Gene Expression

Metabolic reactions are controlled at a number of levels including1) Enzyme activity - fine control rapid (i.e., seconds or less) - posttranslational regulation2) Gene expression - coarser control, slower (minutes) unregulated gene expression - constitutive expression – Genes are continuously expressed at

a constant level. Generally key enzymes and proteins required at about the same level for growth under all conditions are produced in this fashion

regulated gene expression - Enzyme synthesis is adjusted based on cellular needs – regulation can occur at transcriptional or translational levels

Recall: Gene expression = Transcription + Translation

Cells control or regulate gene expression in order to coordinate the numerous metabolic processes in cell Make optimal use of available resources Maintain balance between the amounts of various cellular proteins Adapt to long-term environmental changes Carry out developmental processes

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A Posttranslational Regulation (of Enzyme Activity)

1. Enzyme synthesized as a larger inactive molecule – must be processed into an active form – Examples

Insulin Protein splicing - removal of intein and splicing of the remaining exteins into an

active protein – Mycobacterium leprae DNA gyrase

2. Enzyme molecules are degraded

3. Inhibition of enzyme activity Enzyme is synthesized with full enzymatic activity – activity is reduced or inhibited by

compounds in cell. Feedback inhibition – endproduct of a biosynthetic pathway inhibits the activity of

enzymes earlier in the pathway. Allostery – inhibitor (effector) binds reversibly to allosteric site of enzyme and changes the enzyme conformation so it binds the substrate less efficiently.

4. Modification of enzymes In some bacteria, enzyme activity is regulated by covalent modification of the enzyme.

This changes the conformation of the enzyme Modifying groups include adenylylation (AMP), ADP, Pi or methylation (CH3)

B. Regulation of Transcription

The half-life of a mRNA is usually very short (usually < several minutes in bacteria) Transcription is often regulated by the binding of regulatory proteins to DNA. These

interactions may be nonspecific or specific (sequence specific binding).

1. Nonspecific interactions

Histones are very important in the structure of eukaryotic chromosomes Histones are rich in R, K and H – basic amino acids that are positively charged at

physiological pH DNA associated with histones is unavailable to other proteins such as RNA polymerase

2. Specific interactions Specific interactions usually occur in the major groove of the DNA molecule Inverted repeats are frequently the locations for DNA-protein interactions

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The amount of enzyme produced can be changed at the transcriptional level as well as the translational level. The most obvious way to increase the amount of transcript produced is to increase the strength of a promoter (e.g., promoter sequence is very close to the promoter consensus sequence). High levels of translation can be achieved if the Shine-Dalgarno sequence has extensive complementarity with the 16S rRNA in prokaryotes. These sequences are fixed and serve to set expression at a set level.

What if the cell needs to increase the level of protein expression (i.e., the amount of a particular enzyme)?

The level of transcription can be changed in a regulated manner

i) Negative control of transcription Regulatory mechanisms that prevent initiation of transcription These mechanism prevent wasteful expression of unnecessary enzymes

A common system of negative control involves the use of repressor proteins that inhibit mRNA synthesis. The actions of the specific repressor proteins are under the control of specific small molecules known as effectors

a) Enzyme repression Biosynthetic enzymes are synthesized only if their products are absent in the medium This type of regulation controls the synthesis of a wide variety of enzymes involved in

the biosynthesis of amino acids and nitrogenous bases. In most cases it is the final product of these biosynthetic pathways represses the synthesis

of enzymes involved in this pathway. Corepressor molecule (also known as an effector) represses enzyme production.

Corepressor molecules combine with specific regulatory proteins (repressor) that in turn blocks mRNA synthesis

Specific enzymes are synthesized only when they are needed

Example – repression of the Tryptophan operon (an operon is a cluster of genes, arranged in a linear and consecutive fashion, whose expression is controlled by a single control sequence or operator; Figure 8.13)

Tryptophan combines with the tryptophan repressor protein (an allosteric protein). The conformation of the tryptophan repressor is changed making it capable of

binding to the operator region of the tryptophan operon which between the trp promoter and coding sequences: trpE, trpD, trpC, trpB, and trpA

Synthesis of mRNA is blocked by the bound repressor protein/corepressor complex

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b) Enzyme induction Catabolic enzymes are synthesized only if their substrate is present in the medium This type of regulation is common for enzymes involved in carbon and energy source

catabolism Inducer molecule (also known as an effector) initiates enzyme synthesis. Inducer

molecules combines with specific regulatory proteins (repressor) that in turn initiates mRNA synthesis

Specific enzymes are synthesized only when they are needed

Example – induction of the lactose operon (Figure 8.12) The repressor protein LacI (an allosteric protein) is active in the absence of the

inducer and binds to the operator sequence – blocking mRNA synthesis. When an inducer is added, it binds to LacI. This changes the conformation of

LacI making it incapable of binding to the operator region of the lactose operon. The operator region is between the lac promoter and the coding sequences: lacZ codes for -galactosidase; lacY codes of lactose permease; lacA codes for a transacetylase of unknown function

NOTE: not all effectors are enzyme products or substrates [e.g., Isopropylthiogalactoside (IPTG) induces the lac operon and is not hydrolyzed by -galactosidase]

iii) Positive control of transcription A regulator protein promotes the binding of RNA polymerase thereby acting to increase

the levels of transcription

Example – The maltose regulon Synthesis of maltose catabolic enzymes is controlled at the transcriptional level

by an activator protein The activator protein cannot bind to the DNA unless it first binds maltose (an

effector). When the maltose activator protein binds to the activator binding site of the maltose regulon it allows the RNA polymerase to bind the promoter region and begin transcription.

The sequences of positively controlled promoters are often poor matches for the promoter consensus sequence recognized by the RNA polymerase complex and the activator protein aids the RNA polymerase in promoter recognition or transcription initiation. It may do this by changing the structure of the DNA or interacting directly with the RNA polymerase

The genes required for maltose utilization are found in several different operons, all of which are controlled by the maltose activator protein. More than one operon under the control of the same regulatory protein is known as a regulon.

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3. Global Control SystemsAn organism may need to regulate many different genes in response to an environmental change. This is often accomplished with global control systems

i) Catabolite repressionWhat if Escherichia coli is growing in an environment with several different carbon sources including glucose, lactose and maltose. It would be wasteful to induce enzymes for lactose and maltose catabolism when a better carbon source such as glucose is present. Catabolite repression (a global regulatory network) prevents this problem from occurring. One substrate represses the use of other substates.

Example – the lac operon Catabolite repression ensures that the most readily catabolizable carbon and energy

sources (i.e., glucose) are used first (Recall diauxic growth; Figure 8.14). In E. coli, the enzymes for glucose catabolism are constitutive

Transcription is controlled by an activator protein (a type of positive control) RNA polymerase binds only if a catabolite activator protein (CAP, also known as cAMP-

receptor protein or CRP. It is an allosteric protein) has bound first. CAP binds to the DNA only if it has bound a cyclic AMP (cAMP). CAP binds to a CAP binding site

cAMP is synthesized from ATP by adenylate cyclase Glucose entering the cell inhibits cAMP synthesis and stimulates cAMP transport out of

the cell thus glucose entering the cell reduces the camp levels Catabolite repression is the results of a cAMP deficiency Each of the operons that CAP controls is also under the control of another specific

regulatory protein Transcription of the lac operon requires the presence of lactose and absences of glucose

ii) Other global control systems Often include more than one regulon Modulon – groups of genes that respond to a common global regulatory protein even

though they may also be members of different regulons

a) Alternative sigma factors () Recall that a sigma factor is a RNA polymerase subunit responsible for promoter

recognition, binding and initiation of transcription Different genes may require different sigma factors for RNA polymerase recognition Sigma factor synthesis and activity are controlled by the cell E. coli has 7 different sigma factors referred to by their molecular weight. Most genes

require 70

Sporulation in Bacillus subtilis is controlled by a sigma factor cascade

b) Heat shock response Heat shock genes require 32 to be recognized by the RNA polymerase The amount of 32 in the cell regulates the heat shock response. 32 is normally degraded by proteases in 1 to 2 minutes

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However following heat shock 32 degradation is inhibited and therefore more heat shock proteins are synthesized

c) Quorum sensing Cell density dependent gene expression Cells produce specific acylated homoserine lactones (AHL) that diffuses out of the cell. The AHL is an inducer that combines with an activator and turns on a variety of genes

include those involved in bioluminescence, pigment production, hydrolytic enzyme production and pathogenesis

Activation only occurs when AHL reaches levels that are only achieved above a certain cell concentration (recall that the AHL readily diffuse from the cell)

4. Other mechanisms of regulation

i) Attenuation (to lessen the amount) Control occurs after the initiation of transcription Reduces the number of completed transcripts from a gene or an operon

Example – trp operon trp operon has several levels of regulation: The operon is also under the control of a

repressor as described earlier. Anthranilate synthetase, the first enzyme in the pathway, is also subject to feedback inhibition by Trp (W).

In addition to a promoter and an operator sequence, the trp operon also contains a leader sequence that encodes a polypeptide containing two tandem Trp near the terminus. The leader sequence serves as an attenuator

If Trp is plentiful, then the leader sequence will be synthesized if Trp is lacking, then the Trp rich leader sequence will not be synthesized Synthesis of the leader sequence results in termination of the transcription of the

remainder of the operon – that includes the biosynthetic enzymes for Trp synthesis On the other hand if synthesis of the leader sequence is blocked then transcription of the

remainder of the operon can occur

How does translation of the leader sequence regulate transcription of the downstream sequences? Note: this process cannot occur in eukaryotes! Why?

The process relies on simultaneous transcription and translation Different stem loop structures formed in presence of Trp or absence of Trp result in either

the synthesis of the leader sequence or transcription of the Trp biosynthetic genes. In the presence of Trp a stem-loop transcriptional terminator is formed and only the

leader sequence is translated. If there is little Trp available, the translation of the terminator stalls and a different stem-

loop structure is formed (not a transcriptional terminator) and RNA polymerase transcribes the remainder of the operon.

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Attenuation provides a finer level of control than repression

Attenuation has also been reported in E. coli for biosynthetic pathways for histidine, threonine-isoleucine, phenyalanine and several other amino acids and essential metabolites

5. Signal transduction and two component regulatory systems Bacteria regulate metabolism in response to a wide range of environmental fluctuations

including changes in temperature, pH and oxygen and nutrient availability as well as cell number.

Previous examples involve a signal molecule entering the cell and interacting with the regulatory protein

In other cases, the signal molecule is not transmitted directly to the regulatory protein but rather the signal is detected by a sensor protein which undergoes a change and transmits the signal to the rest of the regulatory machinery – this is know as signal transduction

Two component regulatory systems Characterized by a) specific sensor kinase spanning the cell membrane and b) a

partner response regulator protein (DNA binding protein) The sensor is specific for a specific signal in the environment and in response

phosphorylates itself (autophosphorylation – the sensor is a kinase) at a specific histidine residue.

The phosphoryl group is passed to the response regulator protein which then regulates (i.e., promotes or inhibits) transcription.

Phosphatase activity associated with the sensor kinase or a third protein is required to terminate the signal

V. Genetic Variability

A. Mutations Changes in the genetic makeup (genotype) of a cell Heritable change in the nucleotide sequence of a cell’s DNA alter cell’s genotype - cell’s genetic composition Mutations are an important mechanism for change and production of genetic variation

Designating genotype and phenotypeGenotype Protein PhenotypelacZ LacZ Lac+

hisC HisC His-

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1. Molecular basis of mutations Mutations can be spontaneous or induced. Spontaneous mutants arise without exposure to external

elements and result from replication errors or the action of mobile genetic elements such as transposons. Induced mutations are the result of deliberate exposure to a mutagen.

Types of mutations

i) Mutations involving one or a few base pairs

a) Point mutations - changes in a single nucleotide of a gene

b) If a point mutation is in the polypeptide-coding region of a gene it may have a number of effects

silent mutation -

missense mutation -

nonsense mutation

c) Frame shift mutations - Insertions or deletions - additions or losses of one or more nucleotide pairs in a gene. Usually have dramatic effect because they result in frame shifts or a change in how the template in interpreted.

d) Reversions – back mutations at the same site as the original mutation – produces a revertant with the wild-type phenotype.

e) The mutation restoring wild-type phenotype may occur at a second site. Second site mutations are known as suppressor mutations (new mutation that compensates for the effect of the original mutation)

ii) Mutations involving many base pairs (often not reversible through further mutation)

a) Deletions

b) Insertions – insertion sequences, transposons

c) Translocations – large segments of chromosomal DNA are moved to a new location

d) Inversions – the orientation of a segment of DNA is reversed with respect to surrounding DNA

iii) Mutation Rates1. Errors in replication occur at a frequency of 10-7 to 10-11 therefore in a 1 kb gene error rates

would be between 10-4 to 10-8

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Mutation rates are much higher in RNA genomes (i.e., viruses) because of the lack of RNA repair systems

iv) Detection and Isolation of Mutants

Why study mutations? Knowledge - How can you determine the effect of a trait unless you eliminate it. Development of useful products

e.g., more effective enzymesimproved product production - increased ethanol production

How do we study mutations? Examine the genotype (laborious and time consuming!)

- sequence analysis- restriction analysis

Examine the phenotype using screening (examination of a large number of colonies, looking for different ones) colonial morphology expression of a protein – screen with antibodies (immunodetection) differential media

There are techniques for rapidly screening large numbers of mutants. One technique is replica plating - technique for screening large numbers of colonies on a number of different media. This technique has been used to identify auxotrophic mutants [nutritional mutants that require a growth factor not needed by the parental or wild-type (prototrophic) strain]

If the mutation is selectable (confers an advantage under certain growth conditions), one can use appropriate environmental conditions to detect and isolate selectable mutants. This is known as Positive (direct) selection (e.g., a medium containing an antibiotic). If a mutation can not be selected directly, (i.e., the mutation does not confer a selective advantage) then one can screen indirectly for cells that cannot perform a particular function. This is often done through replica plating (i.e., negative selection) selection screen.

Complementation - restoring wildtype phenotype

Ames test - reversion of Salmonella typhimurium his auxotrophs

2. Mutagens physical and chemical agents which interact with DNA and cause mutations

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i) Chemical agents base analogs (e.g., 2-aminopurine - an analog of guanine and adenine; 5 bromouracil –

thymine analog) incorporated during replication and cause faulty base pairing Modifying agents are chemicals that react with DNA and cause changes even in

nonreplicating DNA e.g., alkylating agents – Nitrosoguanidine (NTG) – adds a methyl group to guanine, causing it to mispair with thymine

intercalating agents (e.g., ethidium bromide, benzopyrene from smoke) – can induce single nucleotide pair insertions and deletions - frame shift mutations during replication

ii) Radiation nonionizing (e.g., UV radiation) – low penetrating power. A common effect is the formation

of pyrimidine dimers that increase the likelihood of errors during replication ionizing (e.g., X-rays, cosmic rays and gamma rays) – produce chemical free radicals that

react with macromolecules damage to DNA. Ionizing radiation may also cause the breakage of the covalent bonds in the sugar-phosphate backbone of nucleic acids

iii) Biological agents Transposable elements – transposon mutagenesis Site-directed mutagenesis

3. DNA Repair (Note: This section is for information only) Microorganisms must be able to changes in DNA sequence that might be lethal Several mechanisms exist besides proofreading by the DNA polymerase. The following is a

description of some of these systems found in E. coli.

i) Excision repair – corrects damage that causes distortion in the DNA double helix Nucleotide excision repair –UvrABC is an endonuclease that removes damaged bases and a

few bases on either side of the lesion. The resulting single stranded ga; (about 12 nt) is filled by DNA polymerase I and the fragments are joined by DNA ligase

Base excision repair – DNA glycosidases remove damaged or unnatural bases. AP endonuclease recognize the damaged DNA and nick the backbone at the AP site. DNA polymerase I removes the damaged region with 5’ to 3’ exonuclease activity and then fills the gap. DNA ligase joins the DNA fragments

ii) Direct Repair – Thymine dimmers and alkylated bases are corrected by direct repair. Photoreactivation uses visible light in a photochemical reaction to thymine dimers; this reaction is catalyzed by photolyase. Methyl and other alkyl groups enzymatically removed by alkyltransferase or methyl guanine methytransferase

iii) Mismatch repair – detect and repair mistakes made during replication. MutS scans newly synthesized DNA for mismatched pairs. MutH removes a stretch of newly synthesized DNA around the mismatch. A DNA polymerase replaces excised nucleotides and DNA ligase joins the fragments

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iv) Recombinational repair – Repairs DNA damage in which both bases are missing or where there is a gap opposite a lesion. The RecA protein cuts a piece of template from a sister molecule and puts it into the gap or uses it to replace the damaged strand.

v) SOS response – sometimes the damage is so great that the normal repair systems cannot repair all of the damage. As a result - DNA synthesis stops completely. This response is made during life and death situations. A global control network called the SOS response is activated. This system relies on RecA

protein. It binds to single stranded or double stranded DNA breaks and gaps resulting from cessation of DNA replication and initiates recombinational repair.

RecA also developes a proteolytic function and destroys a repressor protein known as LexA. LexA negatively regulates the function of many genes involved in DNA repair and synthesis, therefore there is an increase in transcription of genes for excision repair and recombinational repair.

Another regulatory protein, SfiA, is produced and it blocks cell division. If the DNA has not been completed after 40 minutes a process called tranlesional DNA

synthesis is initiated – DNA polymerases IV and V synthesize across gaps that have stopped DNA polymerase III. Incorrect bases may be inserted where intact template is missing. These polymerases also lack proofreading ability and are error prone

B. Genetic Variability and Recombination Exchange of genetic information among different DNA molecules - reshuffling of genetic

information

The most important recombination events in eukaryotes occur during the sexual cycle, including meiosis (e.g., crossing over, segregation of chromosomes into gametes and random fertilization). Genetic variability is transferred vertically from parents to offspring

In contrast, genetic variability in prokaryotes occurs by horizontal gene transfer (HGT, also known as lateral gene transfer). Genes from one independent mature organism are transferred to another). During HGT a piece of DNA must enter and become a stable part of the recipient cell (i.e., it becomes a recombinant). This may happen in one of two ways: 1) the foreign DNA is maintained as an autonomously replicating entity (i.e., it is resistant to the host cell nucleases and is capable of autonomous replication in the host cell), or 2) the foreign DNA is maintained through integration into the host cell genome (i.e., it is not resistant to host cell nucleases and is unable to replicate itself)

Important mechanisms for integration of foreign DNA into the host cell genome

1. Homologous recombination Recombination between homologous DNA sequences (e.g., crossing over between alleles during

prophase I of meiosis)

Reciprocal exchange of genetic material

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2. Site specific recombination The genetic sequence bears only a small region of homology with the chromosome it joins. The

enzymes responsible for this event are often specific for sequences within the particular virus and its host.

e.g., incorporation of phage into bacterial genome

3. Transposition - Transposable elements Elements that can move from one location to another in the cell’s DNA Contain nucleotide sequences that enable them to undergo nonhomologous recombination

Insertions sequences (IS) small 750 -1600 bp repeated regions at ends (inverted repeats - IR) – 15 to 25 bp long and vary among IS carry gene coding transposase - enzyme required for transposition

e.g., IS1, IS2, IS3

Transposons larger than IS elements – include IS sequences (IR and transposase) in addition to other genes Other structural genes commonly include antibiotic resistance factors as well as conjugal genes Designated by Tn followed by an italicized number

e.g., Tn5 - Kmr

Two mechanisms for transposition exist: 1) Simple transposition or “cut and paste” transposition – transposase catalyzed excision followed by cleavage of a new site and ligation of the transposable element into this site or 2) replicative transposition – the transposon remains at the parental site and a replicate is inserted at the target DNA site.

Transposable elements have cause a number of significant effects, including mutations, DNA rearrangments, activation of genes (via transposon promoters) and insertion of plasmids into chromosomes, the spread of antimicrobial resistance factors

Horizontal Gene Transfer There are three important mechanisms for genetic recombination in prokaryotes

1. Transformation Cells take up of naked DNA directly from the environment DNase sensitive DNA is originally derived from donor cell and taken up by recipient which is then called the

transformant Cornerstone technique of molecular biology

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Most bacterial cells will not take up DNA efficiently unless they are exposed to special chemical or electrical treatments. However naturally transformable bacteria can take up DNA from their environment without special treatment

i) Natural competence The naturally transformable bacteria take up DNA when they are in the state of natural

competence. Gram positive and Gram negative bacteria are capable of natural competence

ExamplesBacillus subtilisHaemophilus influenzaeNeisseria gonorrhoeaeStreptococcus pneumoniae (Griffith - 1928 and Avery, McCarty, MacLeod, 1944)

Most naturally transformable bacteria take up DNA at specific growth stages (e.g., late in their growth cycle - usually stationary phase)

A number of genes are involved; B. subtilis - com genes

ii) Artificially induced competence Most bacteria are not naturally transformable, al least nopt at easily detected levels. However this does not preclude them from transformation They can be artificially induced to a state of competence

Methods of Induced Competence

Chemical induction CaCl2 - E. coli Polyethylene glycol - B. thuringiensis

Electroporation formation of transient membrane pores through the use of high voltage fields protocols for many prokaryotic and eukaryotic organisms

2. Transduction genetic material is transferred between donor cell and recipient cell by a viral vectore.g., bacteriophage

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i) Generalized transduction - erroneous packaging of host DNA into viral capsid

Virulent phage and the lytic cycle

ii) Specialized transduction - transmission of specific host genes in the viral genome following imperfect excision during the lysogenic cycle

Temperate phage and lysogenyProphage -Lysogen -

3. Conjugation Directional transfer of DNA from a donor cell to a recipient cell requires physical contact - sex pili often involved This process transfers plasmids and pieces of genome

Conjugal plasmids (F plasmid; RP4; pLS20) Mediate conjugation - carry genetic information necessary for this process

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Conjugal transposons (Tn916 Tcr - spread of drug resistance in Enterococci)

Mobilizable plasmids nonconjugal but transferrable in the presence of a conjugal plasmid

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Principles of Microbial Molecular Biologypeople.uleth.ca/~selibl/Biol3200/CourseNotes/MicroGenCh8... · Web viewProkaryotic DNA fewer proteins associated with it than eukaryotic DNA