Molecular BiologyFourth Edition

Chapter 6

The Mechanism of Transcription in

Bacteria

Lecture PowerPoint to accompany

Robert F. Weaver

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

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6.1 RNA Polymerase StructureBy 1969 SDS-PAGE of RNA polymerase from E. coli had shown several subunits

– 2 very large subunits are (150 kD) and ’ (160 kD)

– Sigma () at 70 kD– Alpha () at 40 kD – 2 copies present in

holoenzyme– Omega (w) at 10 kD

• Was not clearly visible in SDS-PAGE, but seen in other experiments

• Not required for cell viability or in vivo enzyme activity• Appears to play a role in enzyme assembly

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Sigma as a Specificity Factor• Core enzyme without the subunit could not

transcribe viral DNA, yet had no problems with highly nicked calf thymus DNA

• With s subunit, the holoenzyme worked equally well on both types of DNA

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Testing Transcription

• Core enzyme transcribes both DNA strands• Without s-subunit the core enzyme has basic

transcribing ability but lacks specificity

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6.2 Promoters

• Nicks and gaps are good sites for RNA polymerase to bind nonspecifically

• Presence of the -subunit permitted recognition of authentic RNA polymerase binding sites

• Polymerase binding sites are called promoters

• Transcription that begins at promoters is specific, directed by the -subunit

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Binding of RNA Polymerase to Promoters

• How tightly does core enzyme v. holoenzyme bind DNA?

• Experiment measures binding of DNA to enzyme using nitrocellulose filters– Holoenzyme binds filters

tightly– Core enzyme binding is

more transient

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Temperature and RNA Polymerase Binding

• As temperature is lowered, the binding of RNA polymerase to DNA decreases dramatically

• Higher temperature promotes DNA melting

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RNA Polymerase Binding

Hinkle and Chamberlin proposed:• RNA polymerase holoenzyme binds DNA

loosely at first– Binds at promoter initially– Scans along the DNA until it finds one

• Complex with holoenzyme loosely bound at the promoter is a closed promoter complex as DNA is in a closed ds form

• Holoenzyme can then melt a short DNA region at the promoter to form an open promoter complex with polymerase bound tightly to DNA

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Polymerase/Promoter Binding

• Holoenzyme binds DNA loosely at first

• Complex loosely bound at promoter = closed promoter complex, dsDNA in closed form

• Holoenzyme melts DNA at promoter forming open promoter complex - polymerase tightly bound

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Core Promoter Elements

• There is a region common to bacterial promoters described as 6-7 bp centered about 10 bp upstream of the start of transcription = -10 box

• Another short sequence centered 35 bp upstream is known as the -35 box

• Comparison of thousands of promoters has produced a consensus sequence for each of these boxes

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Promoter Strength• Consensus sequences:

– -10 box sequence approximates TAtAaT– -35 box sequence approximates TTGACa

• Mutations that weaken promoter binding:– Down mutations– Increase deviation from the consensus

sequence

• Mutations that strengthen promoter binding:– Up mutations– Decrease deviation from the consensus

sequence

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UP Element

• UP element is a promoter, stimulating transcription by a factor of 30

• UP is associated with 3 “Fis” sites which are binding sites for transcription-activator protein Fis, not for the polymerase itself

• Transcription from the rrn promoters respond – Positively to increased concentration of iNTP– Negatively to the alarmone ppGpp

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The rrnB P1 Promoter

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6.3 Transcription Initiation

• Transcription initiation was assumed to end as RNA polymerase formed 1st phosphodiester bond

• Carpousis and Gralla found that very small oligonucleotides (2-6 nt long) are made without RNA polymerase leaving the DNA

• Abortive transcripts such as these have been found up to 10 nt

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Stages of Transcription Initiation

• Formation of a closed promoter complex

• Conversion of the closed promoter complex to an open promoter complex

• Polymerizing the early nucleotides – polymerase at the promoter

• Promoter clearance – transcript becomes long enough to form a stable hybrid with template

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The Functions of

• Gene selection for transcription by causes tight binding between RNA polymerase and promoters

• Tight binding depends on local melting of DNA that permits open promoter complex

• Dissociation of from core after sponsoring polymerase-promoter binding

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Sigma Stimulates Transcription Initiation

• Stimulation by appears to cause both initiation and elongation

• Or stimulating initiation provides more initiated chains for core polymerase to elongate

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Reuse of

• During initiation can be recycled for additional use in a process called the cycle

• Core enzyme can release which then associates with another core enzyme

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Sigma May Not Dissociate from Core During Elongation

• The s-factor changes its relationship to the core polymerase during elongation

• It may not dissociate from the core

• May actually shift position and become more loosely bound to core

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Fluorescence Resonance Energy Transfer

• Fluorescence resonance energy transfer (FRET) relies on the fact that two fluorescent molecules close together will engage in transfer of resonance energy

• FRET allows the position of relative to a site on the DNA to be measured with using separation techniques that might displace from the core enzyme

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FRET Assay for Movement Relative to DNA

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Local DNA Melting at the Promoter

• From the number of RNA polymerase holoenzymes bound to DNA, it was calculated that each polymerase caused a separation of about 10 bp

• In another experiment, the length of the melted region was found to be 12 bp

• Later, size of the DNA transcription bubble in complexes where transcription was active was found to be 17-18 bp

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Region of Early Promoter Melted by RNA Polymerase

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Structure and Function of

• Genes encoding a variety of -factors have been cloned and sequenced

• There are striking similarities in amino acid sequence clustered in 4 regions

• Conservation of sequence in these regions suggests important function

• All of the 4 sequences are involved in binding to core and DNA

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Homologous Regions in Bacterial Factors

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E. coli 70

• Four regions of high sequence similarity are indicated

• Specific areas that recognize the core promoter elements, -10 box and –35 box are notes

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Region 1

• Role of region 1 appears to be in preventing from binding to DNA by itself

• This is important as binding to promoters could inhibit holoenzyme binding and thereby inhibit transcription

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Region 2

• This region is the most highly conserved of the four

• There are four subregions – 2.1 to 2.4

• 2.4 recognizes the promoter’s -10 box

• The 2.4 region appears to be -helix

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Regions 3 and 4

• Region 3 is involved in both core and DNA binding

• Region 4 is divided into 2 subregions– This region seems to have a key role in

promoter recognition– Subregion 4.2 contains a helix-turn-helix

DNA-binding domain and appears to govern binding to the -35 box of the promoter

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Summary

• Comparison of different gene sequences reveals 4 regions of similarity among a wide variety of sources

• Subregions 2.4 and 4.2 are involved in promoter -10 box and -35 box recognition

• The -factor by itself cannot bind to DNA, but DNA interaction with core unmasks a DNA-binding region of

• Region between amino acids 262 and 309 of ’ stimulates binding to the nontemplate strand in the -10 region of the promoter

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Role of -Subunit in UP Element Recognition

• RNA polymerase itself can recognize an upstream promoter element, UP element

• While -factor recognizes the core promoter elements, what recognizes the UP element?

• It appears to be the -subunit of the core polymerase

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Modeling the Function of the C-Terminal Domain

• RNA polymerase binds to a core promoter via its -factor, no help from C-terminal domain of -subunit

• Binds to a promoter with an UP element using plus the -subunit C-terminal domains

• Results in very strong interaction between polymerase and promoter

• This produces a high level of transcription

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6.4 Elongation

• After transcription initiation is accomplished, core polymerase continues to elongate the RNA

• Nucleotides are added sequentially, one after another in the process of elongation

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Function of the Core Polymerase

• Core polymerase contains the RNA synthesizing machinery

• Phosphodiester bond formation involves the - and ’-subunits

• These subunits also participate in DNA binding

• Assembly of the core polymerase is a major role of the -subunit

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Role of in Phosphodiester Bond Formation

• Core subunit lies near the active site of the RNA polymerase

• This active site is where the phosphodiester bonds are formed linking the nucleotides

• The -factor may also be near nucleotide-binding site during initiation phase

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Role of ’ and in DNA Binding• In 1996, Evgeny Nudler and colleagues

showed that both the - and ’-subunits are involved in DNA binding

• They also showed that 2 DNA binding sites are present– A relatively weak upstream site

• DNA melting occurs• Electrostatic forces are predominant

– Strong, downstream binding site where hydrophobic forces bind DNA and protein together

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Strategy to Identify Template Requirements

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Observations Relating to Polymerase Binding

• Template transfer experiments have delineated two DNA sites that interact with polymerase

• One site is weak– It involves the melted DNA zone, along with

catalytic site on or near -subunit of polymerase

– Protein-DNA interactions here are mostly electrostatic and are salt-sensitive

• Other is strong binding site involving DNA downstream of the active site and the enzyme’s ’- and -subunits

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Structure of the Elongation Complex

• How do structural studies compare with functional studies of the core polymerase subunits?

• How does the polymerase deal with problems of unwinding and rewinding templates?

• How does it move along the helical template without twisting RNA product around the template?

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RNA-DNA Hybrid

• The area of RNA-DNA hybridization within the E. coli elongation complex extends from position –1 to –8 or –9 relative to the 3’ end of the nascent RNA

• In T7 the similar hybrid appears to be 8 bp long

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Structure of the Core Polymerase

• X-ray crystallography on the Thermus aquaticus RNA polymerase core reveals an enzyme shaped like a crab claw

• It appears designed to grasp the DNA

• A channel through the enzyme includes the catalytic center– Mg2+ ion coordinated by 3 Asp residues– Rifampicin-binding site

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Structure of the Holoenzyme

• Crystal structure of T. aquaticus RNA polymerase holoenzyme shows an extensive interface between and - and ’-subunits of the core

• Structure also predicts region 1.1 helps open the main channel of the enzyme to admit dsDNA template to form the closed promoter complex

• After helping to open channel, the s will be expelled from the main channel as the channel narrows around the melted DNA of the open promoter complex

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Additional Holoenzyme Features

• Linker joining regions 3 and 4 lies in the RNA exit channel

• As transcripts grow, they experience strong competition from 3-4 linker for occupancy of the exit channel

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Structure of the Holoenzyme-DNA Complex

Crystal structure of T. aquaticus holoenzyme-DNA complex as an open promoter complex reveals:

– DNA is bound mainly to s-subunit

– Interactions between amino acids in region 2.4 of s and -10 box of promoter are possible

– 3 highly conserved aromatic amino acids are able to participate in promoter melting as predicted

– 2 invariant basic amino acids in s predicted to function in DNA binding are positioned to do so

– A form of the polymerase that has 2 Mg2+ ions

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Topology of Elongation

• Elongation of transcription involves polymerization of nucleotides as the RNA polymerase travels along the template DNA

• Polymerase maintains a short melted region of template DNA

• DNA must unwind ahead of the advancing polymerase and close up behind it

• Strain introduced into the template DNA is relaxed by topoisomerases

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6.5 Termination of Transcription

• When the polymerase reaches a terminator at the end of a gene it falls off the template and releases the RNA

• There are 2 main types of terminators– Intrinsic terminators function with the RNA

polymerase by itself without help from other proteins

– Other type depends on auxiliary factor called , these are -dependent terminators

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Rho-Independent Termination

• Intrinsic or r-independent termination depends on terminators of 2 elements:– Inverted repeat followed immediately by– T-rich region in nontemplate strand of the

gene

• An inverted repeat predisposes a transcript to form a hairpin structure

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Inverted Repeats and Hairpins

• The repeat at right is symmetrical around its center shown with a dot

• A transcript of this sequence is self-complementary– Bases can pair up to

form a hairpin as seen in the lower panel

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Structure of an Intrinsic Terminator

• Attenuator contains a DNA sequence that causes premature termination of transcription

• The E. coli trp attenuator was used to show:– Inverted repeat allows a hairpin to form at transcript end– String of T’s in nontemplate strand result in weak rU-dA

base pairs holding the transcript to the template strand

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Model of Intrinsic Termination

Bacterial terminators act by:• Base-pairing of

something to the transcript to destabilize RNA-DNA hybrid– Causes hairpin to form

• Causing the transcription to pause– Causes a string of U’s to

be incorporated just downstream of hairpin

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Rho-Dependent Termination

• Rho caused depression of the ability of RNA polymerase to transcribe phage DNAs in vitro

• This depression was due to termination of transcription

• After termination, polymerase must reinitiate to begin transcribing again

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Rho Affects Chain Elongation

• There is little effect of on transcription initiation, if anything it is increased

• The effect of on total RNA synthesis is a significant decrease

• This is consistent with action of to terminate transcription forcing time-consuming reinitiation

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Rho Causes Production of Shorter Transcripts

• Synthesis of much smaller RNAs occurs in the presence of compared to those made in the absence

• To ensure that this due to , not to RNase activity of , RNA was transcribed without and then incubated in the presence of

• There was no loss of transcript size, so no RNase activity in

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Rho Releases Transcripts from the DNA Template

• Compare the sedimentation of transcripts made in presence and absence of – Without , transcripts cosedimented with the

DNA template – they hadn’t been released– With present in the incubation, transcripts

sedimented more slowly – they were not associated with the DNA template

• It appears that serves to release the RNA transcripts from the DNA template

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Mechanism of Rho• No string of T’s in the -

dependent terminator, just inverted repeat to hairpin

• Binding to the growing transcript, follows the RNA polymerase

• It catches the polymerase as it pauses at the hairpin

• Releases transcript from the DNA-polymerase complex by unwinding the RNA-DNA hybrid