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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 (s) 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
Basic gene structure; transcription start is +1
Fig. 3.20
6.1 RNA Polymerase Structure
SDS-PAGE of RNA polymerase
(RNAP) from E. coli several
subunits:
 b (150 kD) and b’ (160 kD)
– Sigma (s) at 70 kD
– Alpha (a) at 40 kD –
s5
4
3
2
1 holo
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 RNAP
Pcellulose; Fr A, B, C 6-4
Sigma is a Specificity Factor
• Core enzyme (without s subunit) did not transcribe
viral DNA, yet did transcribe nicked calf thymus DNA;
• Core Transcribes both strands (Fig. 2)
• With s subunit, holoenzyme worked equally well on
both types of DNA
6-5
6.2 Promoters
• Nicks and gaps - sites RNAP binds nonspecifically
• The s-subunit permits recognition of authentic RNAP
binding sites
• RNAP binding sites are promoters
• Transcription from promoters is specific, directed by
s-subunit
6-6
RNA Polymerase Binds to Promoters
 s 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
6-7
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
6-8
Polymerase/Promoter Binding: RPc -> RPo
Hinkle & Chamberlin
Holoenzyme binds DNA
loosely at first
Complex 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
6-9
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
6-10
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
6-11
Very strong promoters have UP Element
ex. Promoter for rRNA gene
• UP element (-40 to -60) stimulates transcription 30X;
binds RNAP
• UP region also 3 binding sites for transcriptionactivator protein Fis, (-60 to -150; an enhancer)
• Transcription from these ribosomal rrn promoters
responds to nucleotides (conc. iNTP)
Fig. 7; rrnB P1
promoter
6-12
1 2 3----
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.
6-13
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 s leaves
6-14
Recall RNA transcripts initiate with NTP (triphosphate);
• 1st nucleotide has g phosphate;
• phosphodiester bonds have only a phosphate
•
Fig. 3.13
Sigma Stimulates Initiation
• Stimulation by s 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;
[g -32P]NTP is initiation (most start A)
6-16
Reuse of s
Figs. 11 and 12
• During initiation s recycled for additional use in process called
the s cycle
• Core enzyme can release s; associates with another core
enzyme
• Red [g -32P]ATP; then RifR core + Rif (green) or –Rif (blue)
6-17
Sigma May Not Actually Dissociate from
Core RNAP During Elongation
• Sigma s-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 s relative to site on
DNA without using separation techniques that might displace
s from core RNAP (Ebright and colleagues)
6-18
FRET Assay for s Movement Relative to DNA
Fig. 13 Predictions FRET.
Fig. 14 FRET expt suggests
sigma does not actually
dissociate from RNAP
6-19
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
6-20
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 6-21
reforming, renders melted region sensitive to nuclease S1. R = RNAP, S = S1
Structure and Function of s
• Genes encoding variety of s-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 = s70
of Bacillus subtilis = s43
(masses kD)
6-22
Homologous Regions in Bacterial s Factors
Fig. 19
E. Coli and
B. subtilis s
factors
6-23
E. coli s70
• Specific areas recognize core promoter elements:
-10 box and –35 box
• Region 1: prevents s 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
6-24
Summary of s and RNAP
• Comparison of different s 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
• s-factor alone cannot bind DNA, but DNA
interaction with core RNAP unmasks DNA-binding
region of s
• RNAP region between amino acids 262 and 309 of
b’ stimulates s binding to nontemplate strand in
-10 region of the promoter
6-25
C-Terminal Domain of a subunit of RNAP can
recognize UP element
• RNA polymerase binds
core promoter via s-factor,
no help from C-terminal
domain of a-subunit
• Binds to promoter UP
element using s plus asubunit C-terminal domain
• Very strong interaction
between polymerase and
promoter produces high
level of transcription
Fig. 26 CTD of a subunit
6-26
DNase footprint shows a subunit of RNAP
can bind UP element
• RNAP binds to promoter
with an UP element
using s plus a-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)
Fig. 6.25
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
 a-phosphate enters
phosphodiester bond
Fig. 3.14
(Fig. 2.9; 3.13)
6-28
Function of Core RNA Polymerase
• Core polymerase contains RNA synthesizing
machinery
• Phosphodiester bond formation involves b- and b’subunits
• These subunits also participate in DNA binding
• Assembly of core RNAP is major role of a-subunit
6-29
Functions of RNAP subunits
Purify subunits – urea denatured,
then renatured
Wild-type and drug-resistant –
(Rifampicin blocks initiation)
Mix in different combinations
Rif-r comes from b subunit
Fig. 6.27
6-30
Role of b in Phosphodiester Bond Formation
• Core subunit b 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 s-factor may be near
nucleotide-binding site during
initiation phase
Fig. 29
6-31
Role of b’ and b in DNA Binding
Nudler lab showed both b- and b’-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
6-32
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?
6-33
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
6-34
Structure of
T.aquaticus
RNAP core (Fig. 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
residues
Rifampicin-binding site
Rif is antibiotic that permits
initiation, not elongation
6-35
Structure of Holoenzyme
• Crystal structure of T. aquaticus RNAP holoenzyme
shows extensive interface between s and the b- and
b’-subunits of core
• Predicts s region 1.1 helps open main channel of
enzyme to admit dsDNA template to form RPc
• After open channel, s expelled from main channel as
channel narrows around melted DNA of the RPo
• Linker joining s regions 3-4 lies in RNA exit channel
• As transcripts grow, have strong competition from s3s4 linker for exit channel -> often abortive transcripts
6-36
Structure of Holoenzyme-DNA Complex
Crystal structure of
T. aquaticus RNAP
in synthetic RPo
complex
Fig. 40
– DNA bound mainly to s-subunit
– Interactions between amino acids in region 2.4 of s and -10
box of promoter
– 3 highly conserved aromatic amino acids participate in
promoter melting
– 2 invariant basic amino acids in s predicted to function in
DNA binding are so positioned
– A form of RNAP that has 2 Mg2+ ions
6-37
Holoenzyme-DNA complex
Fig. 41; RNAP bound to special template resembles RPo form 6-38
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
6-39
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
(r): these are r-dependent terminators
6-40
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’
6-41
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
6-42
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
6-43
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)
6-44
Mechanism of Rho
• No string of T’s in rdependent terminator, just
inverted repeat to hairpin
• Rho loads at upstream
sequence
• Binds to growing transcript,
r follows RNAP
• Rho catches RNAP as it
pauses at hairpin
• Rho releases transcript from
DNA-RNAP complex by
unwinding RNA-DNA hybrid
Fig. 51
6-45
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?
6-46
b. Would T-> mutation in last position be up or down?