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