Chapter 6Chapter 6

The Transcription The Transcription Apparatus of Apparatus of ProkaryotesProkaryotes

RNA Polymerase Structure RNA Polymerase Structure

• The subunit content of an RNA polymerase holoenzyme is ’, , 2, ω and .

’ :160 kD; : 150 kD; : 40 kD; 70: 70 kD ; ω: 10 kD

• 3 regions of conservation: -35, -10 and the length of spacer 17 bp 1 bp

Fig. 6.1

Sigma as a Specificity FactorSigma as a Specificity Factor

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

Promoters Promoters

• The polymerase binding sites, including the transcription initiation sites, are called promoters.

holoenzyme

Corepolymerase

RNase-resistance

Binding of RNA polymerase to Binding of RNA polymerase to PromotersPromoters

• 3H-labeled T7 DNA to bind to E. coli core polymerase (blue) or holoenzyme (red).

• Next they added an excess of unlabeled T7 DNA so that any polymerase that dissociated from the labeled DNA would be likely to re-bind to unlabeled DNA

• Filter the mixtures through NC at various times to monitor the dissociation.

T ½= less than 1 min

T ½= 30- 60 hrs

• High temperature promotes DNA melting (strand separation), this finding is consistent with the notion that tight binding involves local melting of the DNA.

More stable

6-11

Polymerase/Promoter BindingPolymerase/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

SummarySummary

• The sigma-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 complex and is only in the presence of sigma.

Promoter StructurePromoter Structure

• Prokaryotic promoters contain two regions centered at –10 and –35 base pairs upstream of the transcription start site. In general, the more closely regions within a promoter resemble these consensus sequences, the stronger that promoter will be.

enhancer

30 X increase of activation

Transcription Initiation Transcription Initiation

• Carpousis allowed E. coli RNA polymerase to synthesize 32P-labeled RNA in vitro using a DNA containing lac UV5 promoter, heparin to bind any free RNA polymerase

• Heparin: negatively charged polysaccharide that competes with DNA in binding tightly to free RNA polymerase

• Abortive transcripts would be up to 9-10 nt in size.

Lane 1: no DNA; lane 2, ATP only; lane 3-7: ATP with concentrations of CTP, GTP, and UTP increasing by two-fold in each lane.

• Because the heparin in the assay prevented free polymerase from re-associating with the DNA, this result implied that the polymerase was making many small, abortive transcripts without ever leaving the promoter.

• The abortive transcripts up to 9 to 10 nt in size.

Fig. 6.9

The Functions of sigma The Functions of sigma

stimulates initiation, but not elongation, of transcription.

can be re-used by different core polymerases, and the core, not , governs rifampicin sensitivity or resistance.

• Rifampicin: blocks prokaryotic transcription initiation but not elongation.

The incorporation of the [14C]ATP measured bulk RNA synthesis; the incorporation of the -32P nucleotide measured initiation

Even though sigma seems to stimulate both initiation and elongation, it was due to an indirect effect of enhanced initiation

Further experimentFurther experiment

stimulates initiation, but not elongation, of transcription was further demonstrated by the use of

“Rifampicin”( blocks prokaryotic transcription initiation but not elongation). They held the number of RNA chains constant and then use ultracentrifugation to measure the length of the RNA in the presence and absence of sigma.

Experiment demonstrate that sigma can be recycled.

• The key was to run the transcription reaction at low ionic strength, which prevent RNA polymerase core from dissociating from the DNA template at the end of a gene.

The number of RNA chain Constant by allowing a certain amount of initiation to occur and then blocking any further initiation by rifampicin; then add rifampicin-resistant core polymerase

+ rifampicin

- rifampicin

6-25

Reuse of 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

Sigma may not associate from core During Sigma may not associate from core During ElongationElongation

• Fluorescence resonance energy transfer (FRET): two fluorescent molecules close to each other will engage in transfer of resonance energy, and the efficiency of this energy transfer will decrease rapidly as the two molecules move apart.

SummarySummary

• The sigma factor changes its relationship to the core polymerase during elongation, but it may not dissociate from the core. Instead it may just shift position and become more loosely bound to the core.

Local DNA melting at the promoterLocal DNA melting at the promoter

• When A is base-paired with T, the N1 nitrogen of A is hidden in the middle of the double helix and is protected from methylation

• S1 nuclease can cut the DNA at each of the unformed base pairs because these are local single-stranded regions.

Lane R+S+ shows the results when both RNA polymerase ( R) and S1 nuclease (S) were used. On binding to a promoter, RNA polymerase causes the melting of at least 10 bp,

Structure of Sigma Structure of Sigma

• Sigma 70 family: There are four conserved regions in sigma 70 family proteins.

• The best evidence for the functions of these regions shows that sub-regions 2.4 and 4.2 are involved in promoter –10 box and –35 box recognition.

• Region 1: found only in the primary sigmas ( sigma 70 and 43)

• Region 2: most highly conserved sigma region, 2.4: -10 box binding

• Region 3: helix-turn-helix DNA binding domain

• Region 4: 4.2 : -35 box binding

Fig. 6.20

Fig. 6.21

P: lacking the tac promoter

In this experiment contains only the region 4, not region 2.

Because pTac DNA competes much better than P DNA, they concluded that the fusion protein with region 4 can bind to the tac promoter.

The role of the The role of the -subunit in UP element -subunit in UP element recognitionrecognition

• The RNA polymerase -subunit has an independently folded C-terminal domain that can recognize and bind to a promoter’s UP element. This allows very tight binding between polymerase and promoter.

• α subunit response to activator, repressor, elongation factor and transcription factors

-235 polymerase: missing 94 C-terminal amino acid of the subunit

In vitro transcription. What is the conclusion you get from this experiment?

-88: wild type promoter; SUB: irrelevant sequence instead; -41: deletion UP

The bold brackets indicate the footprints in the UP element caused by the -subunit, and the thin bracket indicates the footprint caused by the holoenzyme.

ElongationElongation

Core Polymerase Functions in Elongation Core Polymerase Functions in Elongation

• The role of β in phosphodiester bond formation : The core subunitβ binds nucleotides at the active site of the RNA polymerase where phosphodiester bonds are formed. Rifampicin can block initiation by preventing the formation of that first bond.

• The core subunit β’can bind weakly to DNA by itself in vitro. In fact, both β andβ’bind to DNA as indicated by different experiments.

The affinity-labeling reactions: First, add reagent I to RNA polymerase. The reagent binds covalently to amino groups at the active site. Next, add radioactive UTP, which forms a phosphodiester bond (blue) with the enzyme-bound reagent I. This reaction should occur only at the active site, so only that site becomes radioactively labeled.

Labeled the active site as mentioned above, then separate the polymerase subunits to identify the subunits that compose the active site

Electrostatic interaction ,

Hydrophobic interaction and ’

Termination of TranscriptionTermination of Transcription

• Rho-independent Termination: inverted repeats and Hairpins, a string of Ts in the nontemplate strand

rU-dA have a melting temperature 20 degree lower than rU-rA or rA-dT pairs

An assay for attenuationAn assay for attenuation

• If attenuation works, and transcription terminates at the attenuator, a short 140-nt transcript should be the result.

• When change the string of eight T’s in the nontemplate strand, creating a trp a1419 mutant, attenuation was weakened.

• This result is consistent with the weak rU-dA pairs are important in termination.

TTTTGAA: trp a1419, attenuation weakened IMP: inosine monophosphate, a GMP analogue, weaken base-pairing in the hairpin, I=C weaker than GC pair

The essence of a bacterial terminator The essence of a bacterial terminator is twofoldis twofold

• 1. Base-pairing of something to the transcript to destabilize the RNA-DNA hybrid

• 2. Something that causes transcription to pause• A normal intrinsic terminator satisfies the first

condition by causing a hairpin to form in the transcript, and the second by causing a string of U’s to be incorporated just downstream of the hairpin.

• Rho-dependent Termination: consist of an inverted repeat, which can cause a hairpin to form in the transcript, but no string of Ts.

• Rho affects chain elongation, but not initiation. • Rho causes production of shorter transcripts. • Rho is an RNA helicase, composed of 6 identical

subunits, each subunit has an RNA binding domain and ATPase domain

• Rho releases the RNA product from the DNA template.

Chapter 7Chapter 7

Operons: Fine Control of

Prokaryotic Transcription

TheThe lac lac OperonOperon

• Lactose metabolism in E.coli is carried out by two enzymes, with possible involement by a third. The genes for all three enzymes are clustered together and transcribed together from one promoter, yielding a polycistronic message.

• The lac Operon: It contains three structural genes – genes that code for proteins : -galactosidase (lacZ), galactoside permease (lacY), and galactoside transacetylase (lacA).

• They all are transcribed together on one messager RNA, called a polycistronic message, starting from a single promoter.

• Negative Control of the lac Operon

• Repressor-operator Interactions

• Lac repressor binds to lac operator was demonstrated by filter-binding assay.

The repressor is an allosteric protein: one in which the binding of one molecular to the protein changes the shape of a remote site on the protein and alter its interaction with a second molecule. Inducer: 1st molecule; operator: 2nd molecule

Constitutive mutants had a defect in the gene (lacI)

Constitutive mutant

Because it is dominant only with respect to genes on the same DNA

Because the mutant repressor will bind to operators even in the presence of inducer or of WT repressor

Constitutive and dominant

The mechanism of Repression The mechanism of Repression

• RNA polymerase can bind to the lac promoter in the presence of the repressor. The function of the repressor appears to inhibit the transition from the non-productive synthesis of the abortive transcripts to real, processive transcription.

• Cohen and colleagues labeled lacO-containing DNA with 32P and added increasing amounts of lac repressor

• They assayed binding between repressor and operators by measuring the radioactivity attached to NC.

• Only labeled DNA bound to repressor would attach to NC.

• IPTG: prevents repressor-operator binding.

Assaying the binding between lac operator and lac repressor

Mutant O with low affinity

Wild type operator

Nonsense DNA

1. Incubation of a DNA fragment containing the lac promoter with (lanes 2 and 3) or without (lane 1) lac repressor (LacR).

2. After repressor-operator binding had occurred, they added RNA polymerase. After 20 minutes for OC to form, they added heparin and all components except CTP.

3. Finally, after 5 more minutes, they -32P CTP alone or with the inducer IPTG then wait for 10 minutes for RNA synthesis. The result showed that transcription occurred even when repressor bound to the DNA before polymerase could, repressor did not prevent polymerase from binding and forming an open promoter complexes. ( but the condition is nonphysiological conditions, too much proteins)

Effect of Effect of lac lac repressor on dissociation of repressor on dissociation of RNA polymerase from theRNA polymerase from the lac lac promoterpromoter

• Record made complexes between RNA polymerase and DNA containing the lac promoter-operator region

• They allowed the complexes to synthesize abortive transcripts in the presence of a UTP analog fluorescently labeled.

• As the polymerase incorporates UMP from this analog into transcripts, the labeled pyrophosphate released increases in fluorescence intensity.

( condition likely in vivo)

• The latest evidence supports the repressor, by binding to the operator, blocks access by the polymerase to the adjacent promoter.

Effects of mutations in the three Effects of mutations in the three lac lac operatorsoperators

• WT or mutant lac operon on phage

• Infect and lysogenize E. coli

• Assay for -galactosidase in the presence or absence of IPTG

+IPTG/-IPTG

Positive Control of the Positive Control of the laclac Operon Operon

• It is mediated by a factor called catabolite activator protein (CAP) in conjunction with cyclic AMP, to stimulate transcription.

• Sensed the lack of glucose, increase of cAMP.

• CAP is dimeric and binds to 22 bp operator sequences, accelerates the initiation of transcription at these promoters.

Once the first phosphodiester bond forms, the polymerase is resistant to rifampicin inhibition until it re-initiates.

CAP binding sites in the lac, gal and ara operons all contain the sequence TGTGALac operon has remarkably weak promoter , -35 box

Mechanism of CAP Action Mechanism of CAP Action

• The CAP-cAMP complex stimulates transcription of the lac operon by binding to an activator site adjacent to the promoter and helping RNA polymerase to bind to the promoter. This closed complex then converts to an open promoter complex. CAP-cAMP causes recruitment through protein-protein interactions, by bending the DNA, or by a combination of these phenomena.

Binding of CAP-cAMP to the activator site Binding of CAP-cAMP to the activator site does cause the DNA to benddoes cause the DNA to bend

• When a piece of DNA is bent, it migrates more slowly during electrophoresis.

• The closer the bend is to the middle of the DNA, the more slowly the DNA electrophoreses.

• Actual electrophoresis results with CAP-cAMP and DNA fragments containing the lac promoter at various points in the fragment, dependent on which restriction enzyme was used to cut the DNA.

Fig. 7.19

Fig. 7.20

Tryptophan’s Role in Negative Tryptophan’s Role in Negative Control of theControl of the trp trp Operon Operon

• The trp Operon contains the genes for the enzymes that E. coli needs to make the amino acid tryptophan.

• The trp operon responds to a repressor that includes a corepressor, tryptophan, which signals the cell that it has made enough of this amino acid. The corepressor binds to the aporepressor, changing its conformation so it can bind to the trp operator, thereby repressing the operon.

Fig. 7.28

High conc. of tryptophan is a signal to turn off the operon

5 structural genes

Trp repressor

Control of theControl of the trp trp Operon by Operon by Attenuation Attenuation

• Because of the weak repression of the trp operon, another extra control called attenuation exists.

• Attenuation imposes an extra level of control on an operon, over and above the repressor-operator system. It operates by causing premature termination of transcription of the operon when the operon’s products are abundant.

Figure 7.30 Two Structures available to the leader-attenuator transcript.

RiboswitchesRiboswitches• Is a region in the 5’-UTR of an mRNA that contains

two modules: an aptamer that can bind a ligand, and an expression plateform whose change in conformation can cause a change in expression of the gene.

• FMN can bind to an aptamer in a riboswitch called the RFN element in the 5’-UTR of the ribD mRNA.

• Upon binding FMN, the base pairing in the riboswitch changes to create a terminator that attenuates transcription.

Fig. 7.34

Fig. 7.35