Ch. 6 Mechanism of Transcription in Bacteria (not Archaea)Student learning outcomes:• Explain that the core RNA polymerase (RNAP)

consists of multiple subunits• Explain that sigma specificity factor chooses promoter• Explain the basic features of promoter sequences• Explain the nature of terminators:

intrinsic (rho-independent) and rho-dependent• Appreciate how structural analysis have aided

molecular mechanisms of understanding

6-1

Overview of bacterial transcription:• RNA polymerase (RNAP) + sigma () factor bind

promoter sequences (closed complex RPc)

• RNAP locally melts 10-17 bp of DNA (open RPo)

• Initiation of transcription (first few nucleotides)• Elongation of transcription• Termination and release of transcript

• Important Figures: 1, 3, 5, 6*, 9*, 12, 13, 16, 17, 19, 20, 29, 30, 34, 35, 38, 43, 44

• Review questions: 1, 6, 7, 9, 14, 17, 18, 19, 23, 24, 27, 28, 33, 34; Analyt Q 1, 2, 3 6-2

Fig. 3.20

Basic gene structure; transcription start is +1

6-4

6.1 RNA Polymerase Structure

SDS-PAGE of RNA polymerase(RNAP) from E. coli several subunits:

(150 kD) and ’ (160 kD)– Sigma () at 70 kD– Alpha () at 40 kD – 2 copies present

– Omega (w) at 10 kD • Not required for cell viability or in vivo enzyme activity• role in enzyme assembly

Fig. 1 Purifications RNAPPcellulose; Fr A, B, C

5 4 3 2 1 holo

6-5

Sigma is a Specificity Factor• Core enzyme (without subunit) did not transcribe viral DNA,

yet did transcribe nicked calf thymus DNA;• Core Transcribes both strands (Fig. 2)• With subunit, holoenzyme worked equally well on both types

of DNA

6-6

6.2 Promoters

• Nicks and gaps - sites RNAP binds nonspecifically

• The-subunit permits recognition of authentic RNAP binding sites

• RNAP binding sites are promoters

• Transcription from promoters is specific, directed by -subunit

6-7

RNA Polymerase Binds to Promoters

stimulates tight binding of RNAP to promoter DNA

• Measured binding of T7 DNA to RNAP using nitrocellulose filters– Protein sticks to filter, plus DNA

bound to it;

– At to, add excess unlabeled DNA, replaces labeled if RNAP falls off

– Holoenzyme binds DNA tightly– Core enzyme binding is weak

Fig. 3

6-8

Temperature and RNAP Binding to promoter

• Form complexes, add lots unlabeled DNA

• At lower temperatures, binding of RNAP to T7 DNA is decreased

• Higher temperature promotes DNA melting -> stronger complexes

Fig. 4

6-9

Polymerase/Promoter Binding: RPc -> RPo

Hinkle & ChamberlinHoloenzyme binds DNA

loosely at firstComplex loosely bound at

promoter = closed promoter complex (RPc), dsDNA closed form

Holoenzyme melts DNA at promoter forming open promoter complex – (Rpo) polymerase tightly bound Fig. 5

6-10

Core Promoter Elements are conserved• Region common to bacterial promoters 6-7 bp long, 10 bp upstream of transcription start (+1) = -10 box • Sequence centered 35 bp upstream is -35 box• Comparison of thousands of promoters gave

consensus sequence for each of these boxes– (capital letters >50%; lower case <50%)

Fig. 6

6-11

Promoter Strength: transcription amount; reflects RNAP binding

• Consensus sequences:– -10 box sequence approximates TAtAaT– -35 box sequence approximates TTGACa– Start of transcription is defined as +1

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

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

6-12

Very strong promoters have UP Elementex. Promoter for rRNA gene

• UP element (-40 to -60) stimulates transcription 30X; binds RNAP

• UP region also 3 binding sites for transcription-activator protein Fis, (-60 to -150; an enhancer)

• Transcription from these ribosomal rrn promoters responds to nucleotides (conc. iNTP)

Fig. 7; rrnB P1 promoter

6-13

6.3 Transcription Initiation

• Initiation assumed to end as RNA polymerase formed 1st phosphodiester bond

• Carpousis and Gralla found very small oligonucleotides

(2-6 nt long) made without RNAP leaving DNA

• Abortive transcripts up to 10 nt

Fig. 8; E. coli RNAP; lane 1 no promoter; lane 2 [32P]ATP only; other lanes all nucleotides, inc.

1 2 3----

6-14

Stages of Transcription Initiation

• Formation of closed promoter complex (RPc)

• Conversion of closed promoter complex to open promoter complex (RPo)

• RNAP at promoter -polymerizing early nucleotides

• Promoter clearance –

transcript long enough to form stable hybrid with template

• Factor leaves

Fig. 3.13

• Recall RNA transcripts initiate with NTP (triphosphate); • 1st nucleotide has phosphate; • phosphodiester bonds have only phosphate

6-16

Sigma Stimulates Initiation

• Stimulation by appeared to cause both initiation and elongation

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

• Later expts with rifampicin to block re-initiation showed not elongation

Fig. 10. T4 DNA; [14C]ATP measures bulk RNA; [ -32P]NTP is initiation (most start A)

6-17

Reuse of

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

• Core enzyme can release ; associates with another core enzyme

• Red [ -32P]ATP; then RifR core + Rif (green) or –Rif (blue)

Figs. 11 and 12

6-18

Sigma May Not Actually Dissociate from Core RNAP During Elongation

• Sigma -factor changes its relationship to core RNAP during elongation

• It may not actually dissociate from core• It may shift position and become more loosely bound

• FRET (Fluorescence resonance energy transfer): two fluorescent molecules close together will transfer

resonance energy

FRET permits measurement of position of relative to site on DNA without using separation techniques that might displace from core RNAP (Ebright and colleagues)

6-19

FRET Assay for Movement Relative to DNA

Fig. 13 Predictions FRET.Fig. 14 FRET expt suggests sigma does not actually dissociate from RNAP

6-20

Local DNA Melts at Promoter

• From number of RNAP holoenzymes bound to DNA, calculate each polymerase caused melting of about 10 bp

• In another experiment, length of melted region was about12 bp

• Size of DNA transcription bubble in complexes with active transcription was17-18 bp

• Transcription bubble moves with RNAP, exposing template strand

6-21

Locate region of promoter melted by RNAP: DMS treatment of phage T7 Early Promoter: -9 to +3

Figs. 16, 17: Dimethyl sulfate methylation of DNA prevents base pairs reforming, renders melted region sensitive to nuclease S1. R = RNAP, S = S1

6-22

Structure and Function of

• Genes encoding variety of -factors cloned and sequenced

• Striking similarities in amino acid sequences - clustered in 4 regions

• Conserved sequences suggest important function• All 4 sequences involved in binding RNAP and DNA

• Primary sigmas (routine work): of E. coli = 70 of Bacillus subtilis = 43 (masses

kD)

6-23

Homologous Regions in Bacterial Factors

Fig. 19 E. Coli and B. subtilis factors

6-24

E. coli 70

• Specific areas recognize core promoter elements:

-10 box and –35 box• Region 1: prevents from binding DNA without RNAP• Region 2: very conserved (subregion 2.4

recognizes promoter’s -10 box; alpha helix structure)• Region 3: both RNAP and DNA binding• Region 4: 2 subregions, key role in promoter recognition.

subregion 4.2 has helix-turn-helix DNA-binding domain

binds -35 box of promoter

6-25

Summary of and RNAP

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

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

• -factor alone cannot bind DNA, but DNA interaction with core RNAP unmasks DNA-binding region of

• RNAP region between amino acids 262 and 309 of ’ stimulates binding to nontemplate strand in

-10 region of the promoter

6-26

C-Terminal Domain of subunit of RNAP can recognize UP element

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

• Binds to promoter UP element using plus -subunit C-terminal domain

• Very strong interaction between polymerase and promoter produces high level of transcription Fig. 26 CTD of subunit

Fig. 6.25

DNase footprint shows subunit of RNAP can bind UP element

• RNAP binds to promoter with an UP element using plus -subunit C-terminal domain

• End-labeled template (a) or nontemplate (b) rrnB promoter plus RNAP protein.

• Add DNase; if protein bound, DNase does not cut (footprint)

6-28

6.4 Elongation

• After initiation, core RNAP elongates RNA

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

• Nucleotides enter as triphosphates, but only

-phosphate enters phosphodiester bond

(Fig. 2.9; 3.13)

Fig. 3.14

6-29

Function of Core RNA Polymerase

• Core polymerase contains RNA synthesizing machinery

• Phosphodiester bond formation involves - and ’-subunits

• These subunits also participate in DNA binding

• Assembly of core RNAP is major role of -subunit

Functions of RNAP subunits

6-30Fig. 6.27

Purify subunits – urea denatured, then renaturedWild-type and drug-resistant – (Rifampicin blocks initiation)Mix in different combinationsRif-r comes from subunit

6-31

Role of in Phosphodiester Bond Formation

• Core subunit lies near active site of RNAP:(affinity-label RNAP with ATP analog, then add [32P]UTP and use SDS-PAGE to see which protein subunits are labeled; Figs. 29, 30)

• Active site is where phosphodiester bonds are formed, linking nucleotides

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

Fig. 29

6-32

Role of ’ and in DNA Binding

Nudler lab showed both - and ’-subunits involved in DNA binding: template transfer experiments

Two DNA binding sites :Relatively weak upstream site:

DNA melting occurs

Electrostatic forces

predominant

Strong, downstream site:

hydrophobic forces bind

DNA and protein

Fig. 32 DNA binding sites for RNAP

6-33

Structure of Elongation Complex

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

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

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

6-34

RNA-DNA Hybrids in elongation

• Nudler used RNA-DNA crosslinks (Fig. 34) to measure size of hybrid; special reagent in RNA

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

• In T7 RNAP, similar hybrid appears 8 bp long

Structure of T.aquaticus RNAP core (Fig. 35)

6-35

• X-ray crystallography reveals enzyme shaped like a crab claw: appears designed to grasp the DNA• Channel in RNAP includes catalytic center

Mg2+ ion coordinated by 3 Asp residuesRifampicin-binding site

Rif is antibiotic that permits initiation, not elongation

6-36

Structure of Holoenzyme

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

• Predicts region 1.1 helps open main channel of enzyme to admit dsDNA template to form RPc

• After open channel, expelled from main channel as channel narrows around melted DNA of the RPo

• Linker joining regions 3-4 lies in RNA exit channel

• As transcripts grow, have strong competition from 3-4 linker for exit channel -> often abortive transcripts

6-37

Structure of Holoenzyme-DNA Complex

– DNA bound mainly to -subunit– Interactions between amino acids in region 2.4 of and -10

box of promoter– 3 highly conserved aromatic amino acids participate in

promoter melting– 2 invariant basic amino acids in predicted to function in

DNA binding are so positioned– A form of RNAP that has 2 Mg2+ ions

Crystal structure of T. aquaticus RNAP in synthetic RPo complex Fig. 40

Holoenzyme-DNA complex

6-38Fig. 41; RNAP bound to special template resembles RPo form

6-39

Topology of Elongation• Elongation involves polymerization of nucleotides as

RNAP travels along template DNA• RNAP maintains short melted region of template• DNA must unwind ahead of advancing RNAP and

close up behind it• Strain introduced into template DNA is relaxed by

topoisomerases

Fig. 44 hypotheses for RNAP movement

6-40

6.5 Termination of Transcription

• When RNAP reaches terminator at end of gene, it falls off template and releases RNA

• 2 main types of terminators:– Intrinsic terminators function with RNAP alone

without help from other proteins• Inverted repeat leads transcript to hairpin structure• T-rich region in nontemplate strand produces string of

weak rU-dA base pairs holding transcript to template

– Other type depends on auxiliary factor called Rho (): these are -dependent terminators

6-41

Inverted Repeats and Hairpins

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

• Transcript of sequence is self-complementary

• Bases can pair to form a hairpin (lower panel)

5’

6-42

Structure of an Intrinsic Terminator• Attenuator in trp operon contains DNA sequence

that causes premature termination of transcription• E. coli trp attenuator showed:

– Inverted repeat allows hairpin to form at transcript end– String of T’s in nontemplate strand result in weak rU-dA

base pairs holding transcript to template strand

6-43

Model of Intrinsic Termination

Bacterial terminators :• Base-pairing of something

to transcript destabilizes RNA-DNA hybrid– Causes hairpin to form

• Hairpin causes transcription to pause

• T-rich region nontemplate:– String of U’s incorporated just

downstream of hairpin

6-44

Rho-Dependent Termination

• Rho protein caused decreased ability of RNAP to transcribe phage DNAs in vitro

• Decrease due to termination of transcription• After termination, RNAP must reinitiate to continue

• Rho Affects Chain Elongation (Fig. 48)

• Rho Causes Production of Shorter Transcripts

(Fig. 49)• Rho Releases Transcripts from the DNA Template

(Fig. 50)

6-45

Mechanism of Rho

• No string of T’s in -dependent terminator, just inverted repeat to hairpin

• Rho loads at upstream sequence

• Binds to growing transcript, follows RNAP

• Rho catches RNAP as it pauses at hairpin

• Rho releases transcript from DNA-RNAP complex by unwinding RNA-DNA hybrid

Fig. 51

Review questions

6. Diagram difference between a closed and open promoter complex.

9. Diagram four-step transcription initiation process in E. coli

23. Describe expt to determine which subunit is responsible for rifampicin and streptolydigin resistance or sensitivity.

AQ. An E. coli promoter recognized by RNAP has -10 box in nontemplate strand: 5’-CATAGT-3’.

a. Would C-> T mutation at first position be up or down mutation?

b. Would T-> mutation in last position be up or down?6-46