Molecular BiologyFifth 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 Structure

By 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 subunit, the holoenzyme worked equally well on both types of DNA

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Summary

• The key player in the transcription process is RNA polymerase

• The E. coli enzyme is composed of a core, which contains the basic transcription machinery, and a -factor, which directs the core to transcribe specific genes

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6.2 Promoters

• Why was the core RNA polymerase capable of transcribing nicked DNA in the previous table?

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

• The presence of the -subunit permits recognition of authentic RNA polymerase binding sites 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 the temperature is lowered, the binding of RNA polymerase to DNA decreases dramatically

• Higher temperatures promote DNA melting and encourage RNA polymerase binding

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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 a promoter

• 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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Summary

• The -factor allows initiation of transcription by causing the RNA polymerase holoenzyme to bind tightly to a promoter

• This tight binding depends on local melting of the DNA to form an open promoter complex and is stimulated by

• The -factor can therefore select which genes will be transcribed

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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 (or most common 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

• The UP element is upstream of the core promoter, stimulating transcription by a factor of 30

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

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The rrnB P1 Promoter

• Transcription from the rrn promoters respond

–Positively to increased concentration of iNTP–Negatively to the alarmone ppGpp

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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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Sigma Stimulates Initiation of Transcription

• In this first experiment stimulation by appears to cause both initiation and elongation

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

• Further experiments by the same group proved that does not stimulate elongation

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Reuse of

• During initiation can be recycled for additional use with a new core polymerase

• The core enzyme can release which is then free to associate with another core enzyme

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Fluorescence Resonance Energy Transfer• The -factor changes its relationship to the core

polymerase during elongation• It may not dissociate from the core but actually shift

position and become more loosely bound to core• To answer this question Fluorescence Resonance

Energy Transfer (FRET) was used as it relies on two fluorescent molecules that are close enough together to engage in transfer of resonance energy

• FRET allows the position of relative to a site on the DNA to be measured without 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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Models for the -Cycle

• The obligate release version of the -cycle model arose from experiments performed by Travers and Burgess that proposed the dissociation of from core as polymerase undergoes promoter clearance and switches from initiation to elongation mode

• The stochastic release model proposes that is indeed released from the core polymerase but that there is no discrete point of release during transcription and that the release occurs at random - a preponderance of evidence favors this model

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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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Experiment to locate the region of early promoter melted by RNA Polymerase

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Promoter Clearance

• RNA polymerases have evolved to recognize and bind strongly to promoters

• This poses a challenge when it comes time for promoter clearance as those strong bonds must be broken in order for polymerase to leave the promoter and enter the elongation phase

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Promoter Clearance

• Several hypotheses have been proposed• The polymerase cannot move enough

downstream to make a 10-nt transcript without doing one of three things:

- transient excursion: moving briefly downstream and then snapping back to the starting position- inchworming: stretching itself by leaving its trailing edge in place while moving ots leading edge downstream- scrunching: compressing the DNA without moving itself

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Abortive Transcription, Scrunching and Promoter Clearance

• Ebert and colleagues performed several experiments to distinguish between the hypotheses

• Using E.coli polymerase the authors concluded that approximately 100% of all transcription cycles involved scrunching, which suggested that scrunching is required for promoter clearance

• The E.coli polymerase achieves abortive transcription by scrunching: drawing downstream DNA into the polymerase without actually moving and losing its grip on promoter DNA

• The scrunched DNA could store enough energy to allow the polymerase to break its bonds to the promoter and begin productive transcription

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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 are the -10 box and –35 box

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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

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 (CTD)

• 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 the nucleotide-binding site during the initiation phase

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Structure of the Elongation Complex

• This section will examine how well predictions have been borne out by structural studies

• 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-DNA Complex

Crystal structure of T. aquaticus holoenzyme-DNA

complex as an open promoter complex reveals:

– DNA is bound mainly to -subunit

– Interactions between amino acids in region 2.4 of

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 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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Structure of the Elongation Complex

• The X-ray crystal structure of the Thermus thermophilus RNA polymerase elongation complex in 2007 revealed several important observations– a valine residue in the E’ subunit inserts into

the minor groove of the downstream DNA– the downstream DNA is double-stranded up to

and including the +2 bse pair– the enzyme can accommodate nine base

pairs of RNA-DNA hybrid– the RNA product in the exit channel is twisted

into the shape it would assume as 1/2 of an A-form dsRNA

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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 rewind behind it

• Strain introduced into the template DNA ahead of the transcription bubble is relaxed by topoisomerases

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Pausing and Proofreading

• RNA polymerase frequently pauses, or even backtracks, during elongation

• Pausing allows ribosomes to keep pace with the RNA polymerase, and it is the first step in termination

• Backtracking aids proofreading by extruding the 3’-end of the RNA out of the polymerase, where misincorporated nucleotides can be removed by an inherent nuclease activity of the polymerase, stimulated by auxiliary factors

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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 rho (, these are rho or -dependent terminators

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Rho-Independent Termination

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

gene

• An inverted repeat predisposes a transcript to form a hairpin structure due to complementary base pairing between the inverted repeat sequences

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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

• This causes transcription to pause– a string of U’s incorporated

just downstream of hairpin to destabilize the hybrid and the RNA falls off the DNA template

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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 rho or on transcription initiation, if anything it is increased

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

• This is consistent with action of rho or 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 rho or compared to those made in the absence

• To ensure that this due to itself and 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

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Summary

• Using the trp attenuator as a model rho-independet

terminator revealed two important features:

1 - an inverted repeat that allows a hairpin to for at the

end of the transcript

2 - a string of T’s in the nontemplate strand that results

in a string of weak rU-dA base pairs holding the

transcript to the template strand

• Rho-dependent terminators consist of an inverted

repeat, which can cause a hairpin to form in the

transcript but no string of T’s