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
• 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.
10.2 Mechanism of transcription
• 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'.
• 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.
• Minimal requirements for gene transcription.
– Gene promoter
– RNA polymerase
• Additional factors are required for the regulation of transcription.
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.
• 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.
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
• 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 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
• 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.
• 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
• 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 consists of three stages:
1.Formation of a closed promoter complex.
2.Formation of an open promoter complex.
3.Promoter clearance.
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.
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.
• Transcription is initiated in the presence of NTPs.
• No primer is required for initiation by RNA polymerase.
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
• After about 9-12 nt of RNA have been synthesized, the initiation complex enters the elongation stage.
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.
• 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.
• 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.
• 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.
• 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?
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.
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.
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 by RNA polymerase
• Backtracks 3′→5′
• Pauses
• Nucleolytic cleavage
Termination of transcription
In most bacteria, there are two types of terminators:
• Rho-independent
• Rho-dependent
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
• 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.
• 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.
10.3 Insights into gene regulation from the lactose (lac) operon
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
• 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
• 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
• 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
• 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.
• 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.
• Bacteria need to respond swiftly to changes in their environment, switching from metabolizing one substrate to another quickly and efficiently.
• Induction is the synthesis of enzymes in response to the appearance of a specific substrate.
• When provided with a mixture of sugars, bacteria use glucose first.
Unifying theme in gene transcription
• A regulatory protein (trans-acting factor) binds to a particular sequence of DNA (cis-acting factor)
Trans-acting factor gene
Transcription
Translation
Gene coding regionCis-acting DNA
sequence
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.
• 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.
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
• The basal level of transcription is determined by the frequency with which RNA polymerase spontaneously binds the promoter and initiates transcription.
• 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.
• 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
• 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
• 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.
10.4 Mode of action of transcriptional regulators
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.
• 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.
• 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
CAP has two major functional domains
• DNA-binding domain
• “Activating” domain which contacts RNA polymerase
• 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
• 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.
• 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.
• 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.
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.
• 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 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.
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.
10.5 Control of gene expression by RNA
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
Regulation of the tryptophan operon occurs by two mechanisms:
• Transcriptional attenuation.
• Conventional protein-mediated repression.
• 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.
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.
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.
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.
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
• 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.”
• Widespread in bacteria.
• In eukaryotes, only one type of riboswitch found so far in plants and fungi.
• 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
• 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.
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.
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”
• 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
• 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.
10.6 Gene regulatory networks
• 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
• 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.
• 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.
• 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
• 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.
• 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.
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.
• 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 the luciferase operon.
• The luciferase operon is activated.
• The luxAB genes encode the subunits of luciferase, the enzyme required for light production.
• luxCDE encodes a complex that produces and recycles the substrate.
Top Related