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.