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Chapter 10: Transcription in Bacteria

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Chapter 10: Transcription in Bacteria. Few proteins have had such strong impact on a field as the lac repressor has had in Molecular Biology. Michael Lewis, Comptes Rendus Biologies (2005), 328:521. 10.1 Introduction.

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slide1
Chapter 10:

Transcription in Bacteria

slide2
Few proteins have had such strong impact on a field as the lac repressor has had in Molecular Biology.

Michael Lewis, Comptes Rendus Biologies (2005), 328:521

slide4
A central event in gene expression is the copying of the sequence of the template strand of a gene into a complementary RNA transcript.
  • The biochemistry of transcript formation is straightforward.
  • The regulatory mechanisms that have been developed by bacteria to control transcription are complex and highly variable.
slide6
RNA polymerase is the enzyme that catalyzes RNA synthesis.
  • Using DNA as a template, RNA polymerase joins, or “polymerizes,” nucleoside triphosphates (NTPs) by phosphodiester bonds from 5' to 3'.
slide7
In bacteria, transcription and translation are coupled―they occur within a single cellular compartment.
  • As soon as transcription of the mRNA begins, ribosomes attach and initiate protein synthesis.
  • The whole process occurs within minutes.
slide8
Minimal requirements for gene transcription.
    • Gene promoter
    • RNA polymerase
  • Additional factors are required for the regulation of transcription.
bacterial promoter structure
Bacterial promoter structure
  • RNA polymerase binds to a region of DNA called a promoter.
  • Bacterial promoters are not absolutely conserved but they do have a consensus sequence.
slide10
Conserved sequence: When nucleotide sequences of DNA are aligned with each other, each has exactly the same series of nucleotides in a given region.
  • Consensus sequence: there is some variation in the sequence but certain nucleotides are present at high frequency.
slide11
Promoter strength
  • The relative frequency of transcription initiation.
  • Related to the affinity of RNA polymerase for the promoter region.
  • The more closely regions within the promoter resemble the consensus sequences, the greater the strength of the promoter.
structure of bacterial rna polymerase
Structure of bacterial RNA polymerase
  • Comprised of a core enzyme plus a transcription factor called the sigma factor ().
  • Together they form the complete, fully functional enzyme complex called the holoenzyme.
the core enzyme
The core enzyme
  • The core enzyme catalyzes polymerization.
  • High affinity for most DNA.
  • The sequence, structure, and function are evolutionarily conserved from bacteria to humans.
  • X-ray crystallographic studies revealed a crab claw-like shape.
sigma factor
Sigma factor
  • The sigma () factor decreases the nonspecific binding affinity of the core enzyme.
  • Binding results in closing of the core enzyme “pincers.”
  • Primarily involved in recognition of gene promoters.
slide15
In E. coli the most abundant  factor is 70.
  • For expression of some genes, bacterial cells use alternative  factors.
the sigma factor stimulates tight binding of rna polymerase to the promoter
The sigma factor stimulates tight binding of RNA polymerase to the promoter
  • Shown over 30 years ago using a nitrocellulose filter binding assay.
  • The holoenzyme containing  dissociates more slowly from template DNA compared with the core polymerase alone.
initiation of transcription
Initiation of transcription

Initiation consists of three stages:

  • Formation of a closed promoter complex.
  • Formation of an open promoter complex.
  • Promoter clearance.
slide18
Closed promoter complex
  • RNA polymerase holoenzyme binds to the promoter at nucleotide positions 35 and 10.
  • The DNA remains double-stranded.
  • The complex is reversible.
slide19
Open promoter complex
  • ~18 bp around the transcription start site are melted to expose the template strand DNA.
  • AT rich promoters require less energy to melt.
  • Transcription is aided by negative supercoiling of the promoter region of some genes.
  • The open complex is generally irreversible.
slide20
Transcription is initiated in the presence of NTPs.
  • No primer is required for initiation by RNA polymerase.
promoter clearance
Promoter clearance

Older “classic” model

  •  factor release.

Current model

  •  factor does not completely dissociate; some domains are displaced.
  • The displaced domains allow the nascent RNA to emerge from the RNA exit channel.
elongation
Elongation
  • After about 9-12 nt of RNA have been synthesized, the initiation complex enters the elongation stage.
slide23
Direction of transcription around the E. coli chromosome
  • Of the 50 operons or genes whose transcription direction is known, 27 are transcribed clockwise and 23 in the counterclockwise direction around the circle, using the opposite strand as a template.
  • Only one strand of a given operon’s DNA is used as a template for transcription.
slide24
The origin and terminus of replication divide the genome into oppositely replicated halves or “replichores.”
  • Most operons or genes are transcribed in the direction of replication.
  • This may lead to fewer collisions of DNA and RNA polymerase and less topological strain from opposing supercoils.
slide25
As RNA polymerase moves during elongation, it holds the DNA strands apart, forming a transcription “bubble.”
  • The moving polymerase protects a “footprint” of ~30 bp along the DNA against nuclease digestion.
slide26
One strand of DNA acts as the template for RNA synthesis by complementary base pairing.
  • The catalytic site has both a substrate-binding and a product-binding site.
  • Transcription always proceeds in the 5′→3′ direction.
slide27
Completion of the single nucleotide addition cycle.
  • Shift of the active site of the RNA polymerase by one position along the template DNA.
which moves the rna polymerase or the dna
Which moves – the RNA polymerase or the DNA?

Two models

  • Model 1: RNA polymerase moves along and the DNA rotates.
    • This is the more widely accepted model.
  • Model 2:RNA polymerase remains stationary, and the DNA moves along and rotates.
slide29
The overall process of transcription has a significant local effect on DNA structure
  • The DNA ahead of the RNA polymerase is wound more tightly; positive supercoils form.
  • Behind the polymerase, DNA becomes less tightly wound; negative supercoils form.
  • Topoisomerase I and gyrase (bacterial topoisomerase II) resolve this supercoiling and restore the DNA to its relaxed form.
slide30
RNA polymerase is a molecular motor
  • RNA polymerase rotates the DNA.
  • Tracks the DNA helix over thousands of base pairs, producing measurable torque.
  • A real-time optical microscopy in vitro assay was used to catch RNA polymerase in the act of transcribing.
proofreading
Proofreading

Proofreading by RNA polymerase

  • Backtracks 3′→5′
  • Pauses
  • Nucleolytic cleavage
termination of transcription
Termination of transcription

In most bacteria, there are two types of terminators:

  • Rho-independent
  • Rho-dependent
rho independent termination
Rho-independent termination
  • Terminator is characterized by an inverted repeat consensus sequence.
  • Formation of a stem-loop in the exit channel.
  • Less stable U-A hybrid helix.
  • Polymerase pauses, resulting in transcript release.
rho dependent termination
Rho-dependent termination
  • Terminator is an inverted repeat with no simple consensus sequence.
  • Controlled by the ability of the Rho protein to gain access to the mRNA.
  • Because ribosomes translate mRNA at the same rate as the mRNA is transcribed, Rho is prevented from loading onto the newly formed RNA until the end of a gene or operon.
slide35
Rho binds specifically to a C-rich site called a Rho utilization (rut) site.
  • ATP-dependent polymerase “chasing.”
  • Polymerase pauses at the terminator stem-loop structure, Rho catches up and unwinds the DNA-RNA hybrid.
  • Transcript release.
slide37
The 1959 operon model of Jacob and Monod
  • Novel concept of regulatory genes that code for products that control other genes.
  • Model predicted the existence of an unstable RNA as an intermediate in protein synthesis.
the jacob monod operon model of gene regulation
The Jacob-Monod operon model of gene regulation
  • Model arose from experimental observations in bacteria and phages.
  • Study of how phage lambda () can be induced to switch from lysogenic to lytic state.
  • Study of how the enzyme -galactosidase is produced in bacterial cells only when bacteria need this enzyme to use the sugar lactose
slide39
The lac operon provides an example of negative control of the enzymes involved in lactose metabolism.
  • The lac operon is also regulated by positive control under certain environmental conditions.
characterization of the lac repressor
Characterization of the Lac repressor
  • Jacob-Monod model also proposed the existence of a repressor protein.
  • Gilbert and Müller-Hill isolated the Lac repressor and demonstrated that it binds operator DNA.
lactose lac operon regulation
Lactose (lac) operon regulation
  • In bacteria, genes are organized into operons.
  • An operon is a unit of bacterial gene expression and regulation, including structural genes and control elements in DNA recognized by regulatory gene product(s).
  • Transcribed from a single promoter to produce a single primary transcript of polycistronic mRNA.
slide42
In eukaryotes, genes are not typically organized into operons.
  • Exception
    • ~15% of genes in Caenorhabditis elegans are grouped into operons.
    • But, each C. elegans pre-mRNA is processed into a separate mRNA for each gene rather than being translated as a unit.
slide43
Bacteria need to respond swiftly to changes in their environment, switching from metabolizing one substrate to another quickly and efficiently.
  • Inductionis the synthesis of enzymes in response to the appearance of a specific substrate.
  • When provided with a mixture of sugars, bacteria use glucose first.
slide44
Cis-acting DNA sequence

Trans-acting factor gene

Gene coding region

Transcription

Translation

Unifying theme in gene transcription

  • A regulatory protein (trans-acting factor) binds to a particular sequence of DNA (cis-acting factor)
lac operon induction
Lac operon induction
  • The lac operon consists of three structural genes, lacZ, lacY, and lacA.
  • lacZ encodes -galactosidase, an enzyme which cleaves lactose into galactose and glucose.
  • lacY encodes a lactose permease, part of the transport system to bring lactose into the cell.
  • lacA encodes a transacetylase that rids the cell of toxic thioglactosides that get taken up by the permease.
slide46
In the absence of lactose, the Lac repressor binds the operator and excludes RNA polymerase.
  • In the presence of lactose the lac operon is induced.
  • Recruitment of RNA polymerase requires formation of a complex of the cAMP-bound activator protein CAP, polymerase, and DNA.
slide47
Allolactose – the real inducer
  • The real inducer of the lac operon is an alternative form of lactose called allolactose.
  • When -galactosidase cleaves lactose, it rearranges a small fraction of the lactose to allolactose.
basal transcription of the lac operon
Basal transcription of the lac operon
  • The basal level of transcription is determined by the frequency with which RNA polymerase spontaneously binds the promoter and initiates transcription.
slide49
The lac operon is transcribed if and only if lactose is present in the medium.
  • But, this signal is almost entirely overridden by the simultaneous presence of glucose.
slide50
Glucose exerts its effect, in part, by decreasing synthesis of cAMP which is required for the activator CAP to bind DNA.
  • More importantly, however, glucose inactivates the lactose permease.
  • Without cooperative binding of CAP, RNA polymerase transcribes the lac genes at low level.
regulation of the lac operon by rho
Regulation of the lac operon by Rho
  • When cells are starved of amino acids, a Rho-dependent terminator stops synthesis of mRNA.
the lac promoter and lacz structural gene are widely used in molecular biology research
The lac promoter and lacZ structural gene are widely used in molecular biology research
  • Commonly used reporter gene.
  • Expression of heterologous proteins in bacteria.
  • In the lab IPTG is used as an inducer; it interacts with the Lac repressor but is not metabolized by -galactosidase.
slide54
The lac operon and other operons illustrate fundamental principles of gene regulation that are universal.
  • Constitutively active RNA polymerase that alone works with a certain frequency.
  • Transcriptional activators increase the frequency of initiation.
  • Transcriptional repressors decrease the frequency of initiation.
slide55
The repressors and activators are DNA-binding proteins that undergo allosteric modification.
  • Transcriptional repressors and activators are modular proteins that have domains with distinct functions, e.g for DNA binding, ligand binding, and protein-protein interactions.
slide56
Cooperative binding of proteins to DNA increases their effective binding constants and allows regulatory proteins to function at very low concentrations within the cell.
  • DNA looping allows multiple proteins to interact with RNA polymerase, from adjacent and distant sites.
cooperative binding of proteins to dna
Cooperative binding of proteins to DNA

CAP has two major functional domains

  • DNA-binding domain
  • “Activating” domain which contacts RNA polymerase
slide58
CAP recruits RNA polymerase to the promoter.
  • Helps RNA polymerase bind tightly to the promoter until the polymerase changes from the closed to open complex.
  • One protein might dissociate from the DNA, but due to its continued interaction with the other DNA-bound protein, it does not diffuse away and is more likely to rebind to its DNA site.
allosteric modification and dna binding
Allosteric modification and DNA binding
  • Both CAP and the Lac repressor bind to their DNA sites using a helix-turn-helix motif.
  • Predominant DNA recognition motif found among E. coli transcriptional regulatory proteins.
  • The recognition helix inserts into the major groove of the DNA and make sequence-specific contacts with exposed base pairs.
slide60
Allosteric change undergone by CAP upon binding DNA increases its ability to bind DNA.
  • Allosteric change in the Lac repressor upon binding allolactose decreases its ability to bind DNA.
slide61
The Lac repressor functions as a “dimer of dimers”
  • When bound to allolactose (or IPTG) the helix-turn-helix DNA binding motifs become disordered and dissociate from the binding site.
slide62
Interaction of the Lac repressor with lac operator DNA
  • First, binds nonspecifically to DNA.
  • The hinge region remains unstructured.
  • Then, moves by a “random walk” along the DNA.
slide63
Finally, the Lac repressor binds specifically to the lac operator DNA sequence.
  • The hinge region forms an -helix and the DNA bends by ~36.
dna looping
DNA looping
  • DNA looping allows multiple proteins to interact with RNA polymerase, some from adjacent sites and some from distant sites.
  • The Lac tetramer binds to an upstream auxiliary operator and the primary operator DNA sequence forming a DNA loop in between.
  • A classic example of DNA looping is found in the arabinose operon.
slide65
The arabinose operon
  • The regulatory protein AraC acts both as a repressor and activator of transcription.
  • Helical-twist experiments were used to confirm DNA looping of the arabinose operon.
  • Addition of half-integral turns to the arabinose operon sequence interfered with protein-protein interactions and blocked loop formation.
slide67
RNA frequently plays a direct role in controlling gene expression
  • Differential folding of RNA.
  • Riboswitches.
differential folding of rna transcriptional attenuation of the tryptophan operon
Differential folding of RNA: transcriptional attenuation of the tryptophan operon

Regulation of the tryptophan operon occurs by two mechanisms:

  • Transcriptional attenuation.
  • Conventional protein-mediated repression.
slide69
Newly synthesized RNA can fold to form either of two competing hairpin structures:
    • antiterminator or terminator
  • The leader RNA preceding the antiterminator contains a 14 nt coding region, trpL, which includes two tryptophan codons.
slide70
When bacterial cells have adequate levels of tryptophan-charged tRNATrp
  • The leader peptide (trpL) is synthesized.
  • The terminator forms in the leader transcript.
  • Transcription is terminated.
slide71
When cells are deficient in charged tRNATrp
  • The ribosome translating trpL stalls at one of the tryptophan codons.
  • The antiterminator forms and termination is blocked.
  • The structural genes involved in tryptophan biosynthesis are transcribed.
slide72
Conventional protein-mediated repression of the trp operon
  • In the absence of tryptophan, the genes encoding enzymes for the biosynthesis of tryptophan are transcribed and translated.
slide73
When enough tryptophan has been produced:
  • Tryptophan binds to the dimeric Trp repressor protein.
  • The Trp repressor binds the trp operator and blocks access of RNA polymerase to the trp promoter.
riboswitches
Riboswitches
  • Specialized domains within certain mRNAs act as switchable “on-off” elements that selectively bind metabolites and control gene expression without the need for protein transcription factors.
  • Metabolite sensors and “RNA thermometers.”
slide75
Widespread in bacteria.
  • In eukaryotes, only one type of riboswitch found so far in plants and fungi.
slide76
Typically found in the 5′ untranslated region (UTR) of mRNAs.
  • Two main structural domains:
    • Aptamer: RNA receptor that binds a metabolite.
    • Expression platform: converts metabolite-binding events into changes in gene expression via changes in RNA folding.
metabolite sensors
Metabolite sensors
  • Binding of the metabolite usually serves as an “off” switch, decreasing the expression of the gene products used to make the metabolite.
  • Repression occurs either by terminating transcription or by preventing translation initiation.
  • In some rare cases, the metabolite acts as an “on” switch.
slide78
Lysine riboswitch
  • In the absence of lysine, mRNA forms an antiterminator hairpin and transcription proceeds.
  • In the presence of lysine, the aptamer-sensing domains binds lysine triggering formation of a transcription terminator.
slide79
Type III S-adenosylmethionine (SAM) riboswitch
  • In the absence of SAM, the Shine-Dalgarno sequence is accessible for ribosome binding and translation initiation.
  • In the presence of SAM, the Shine-Dalgarno sequence is base-paired in a stem structure, preventing ribosome binding.
rna thermometers
RNA “thermometers”
  • Expression of heat shock genes in root nodule bacteria is regulated by a conserved RNA sequence element called ROSE.
  • At low temperature, translation is prevented by an extended RNA secondary structure.
  • At high temperature, the secondary structure partially melts allowing ribosome access to the mRNA.
riboswitch ribozymes
Riboswitch ribozymes
  • The glmS gene in Bacillus subtilis encodes an enzyme which generates glucosamine-6-phosphate (GlcN6P) from fructose-6-phosphate and glutamine.
  • glmS mRNA is a highly substrate-specific GlcN6P-responsive ribozyme.
  • Self-destruction of the glmS mRNA inhibits further production of GlcN6P.
slide83
Bacterial regulatory networks have evolved to respond with remarkable precision to environmental changes.
  • Alternative sigma factors coordinate the expression of different sets of genes or operons.
  • Bacteria communicate with each other through the production of autoinducers.
alternative sigma factors
Alternative sigma factors
  • In general, organisms with more varied lifestyles have more  factors.
  • The number of  factors varies from 1 in Mycoplasma genitalia to more than 63 in Streptococcus coelicolour.
slide85
E. coli uses 7 alternative  factors to respond to some environmental changes:
    • expression of heat-shock proteins
    • expression of flagellar genes
  • Bacillus subtillis has 18 different  factors, 5 of which regulate the process of sporulation.
slide86
In Borrelia burgdorferi, the Lyme disease spirochete carried by ticks, the alternative sigma factor N stimulates transcription of the rpoS gene encoding the sigma factor S.
  • Activation of N enhances the expression of a large number of N and S genes that lead to virulence.
quorum sensing
Quorum sensing
  • Bacteria communicate through the production of diffusible signal molecules termed autoinducers.
  • These molecules are produced at basal levels and accumulate during growth.
  • Once a critical concentration has been reached, autoinducers can activate or repress a number of target genes for collective responses.
slide88
These responses can include light production, biofilm formation, or virulence.
  • Because the control of gene expression by autodinducers is cell-density-dependent, this phenomenon has been called quorum sensing.
slide89
The LuxIR-type quorum sensing system
  • LuxI and LuxR are essential for control of bioluminescence in Vibrio fischeri.
  • These marine bacteria colonize the light organ of the Hawaiian bobtail squid.
  • LuxI catalyzes synthesis of the autoinducer, 3OC6HSL.
slide90
When the autoinducer accumulates above threshhold, it binds the cytoplasmic receptor LuxR.
  • When 3OC6HSL is bound to LuxR, the complex binds to a DNA regulatory sequence (lux box) upstream of theluciferaseoperon.
  • Theluciferase operon is activated.
slide91
The luxAB genes encode the subunits of luciferase, the enzyme required for light production.
  • luxCDE encodes a complex that produces and recycles the substrate.
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