Transcript Inquiry into Life Twelfth Edition

Lecture PowerPoint to accompany
Molecular Biology
Fourth Edition
Robert F. Weaver
Chapter 6
The Mechanism of
Transcription in
Bacteria
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
6.1 RNA Polymerase Structure
By 1969 SDS-PAGE of RNA polymerase from E.
coli had shown several subunits
– 2 very large subunits are b (150 kD) and b’ (160
kD)
– Sigma (s) at 70 kD
– Alpha (a) 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
6-2
Sigma as a Specificity Factor
• Core enzyme without the s 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
6-3
Testing Transcription
• Core enzyme transcribes both DNA strands
• Without s-subunit the core enzyme has basic
transcribing ability but lacks specificity
6-4
6.2 Promoters
• Nicks and gaps are good sites for RNA
polymerase to bind nonspecifically
• Presence of the s-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 s-subunit
6-5
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
6-6
Temperature and RNA
Polymerase Binding
• As temperature is
lowered, the binding
of RNA polymerase to
DNA decreases
dramatically
• Higher temperature
promotes DNA
melting
6-7
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
6-8
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
6-9
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
6-10
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
6-11
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
6-12
The rrnB P1 Promoter
6-13
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
6-14
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
6-15
The Functions of s
• Gene selection for transcription by s
causes tight binding between RNA
polymerase and promoters
• Tight binding depends on local melting of
DNA that permits open promoter complex
• Dissociation of s from core after
sponsoring polymerase-promoter binding
6-16
Sigma Stimulates Transcription
Initiation
• Stimulation by s appears
to cause both initiation
and elongation
• Or stimulating initiation
provides more initiated
chains for core
polymerase to elongate
6-17
Reuse of s
• During initiation s can be recycled for additional
use in a process called the s cycle
• Core enzyme can release s which then
associates with another core enzyme
6-18
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
6-19
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 s relative to a
site on the DNA to be measured with using
separation techniques that might displace
s from the core enzyme
6-20
FRET Assay for s Movement
Relative to DNA
6-21
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
6-22
Region of Early Promoter
Melted by RNA Polymerase
6-23
Structure and Function of s
• Genes encoding a variety of s-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
6-24
Homologous Regions in
Bacterial s Factors
6-25
E. coli s70
• Four regions of high
sequence similarity
are indicated
• Specific areas that
recognize the core
promoter elements,
-10 box and –35 box
are notes
6-26
Region 1
• Role of region 1 appears to be in
preventing s from binding to DNA by itself
• This is important as s binding to
promoters could inhibit holoenzyme
binding and thereby inhibit transcription
6-27
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 a-helix
6-28
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
6-29
Summary
• Comparison of different s 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 s-factor by itself cannot bind to DNA, but
DNA interaction with core unmasks a DNAbinding region of s
• Region between amino acids 262 and 309 of b’
stimulates s binding to the nontemplate strand in
the -10 region of the promoter
6-30
Role of a-Subunit in UP
Element Recognition
• RNA polymerase itself can recognize an
upstream promoter element, UP element
• While s-factor recognizes the core
promoter elements, what recognizes the
UP element?
• It appears to be the a-subunit of the core
polymerase
6-31
Modeling the Function of the CTerminal Domain
• RNA polymerase binds to a
core promoter via its s-factor,
no help from C-terminal
domain of a-subunit
• Binds to a promoter with an
UP element using s plus the
a-subunit C-terminal
domains
• Results in very strong
interaction between
polymerase and promoter
• This produces a high level of
transcription
6-32
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
6-33
Function of the Core Polymerase
• Core polymerase contains the RNA
synthesizing machinery
• Phosphodiester bond formation involves
the b- and b’-subunits
• These subunits also participate in DNA
binding
• Assembly of the core polymerase is a
major role of the a-subunit
6-34
Role of b in Phosphodiester
Bond Formation
• Core subunit b lies near the active site of
the RNA polymerase
• This active site is where the
phosphodiester bonds are formed linking
the nucleotides
• The s-factor may also be near
nucleotide-binding site during initiation
phase
6-35
Role of b’ and b in DNA Binding
• In 1996, Evgeny Nudler and colleagues
showed that both the b- and b’-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
6-36
Strategy to Identify Template
Requirements
6-37
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 b-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 b’- and b-subunits
6-38
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?
6-39
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
6-40
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
6-41
Structure of the Holoenzyme
• Crystal structure of T. aquaticus RNA
polymerase holoenzyme shows an extensive
interface between s and b- and b’-subunits of
the core
• Structure also predicts s 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
6-42
Additional Holoenzyme
Features
• Linker joining s regions 3 and 4 lies in the
RNA exit channel
• As transcripts grow, they experience
strong competition from s3-s4 linker for
occupancy of the exit channel
6-43
Structure of the HoloenzymeDNA 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
6-44
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
6-45
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
r, these are r-dependent terminators
6-46
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
6-47
Inverted Repeats and Hairpins
• The repeat at right is
symmetrical around
its center shown with
a dot
• A transcript of this
sequence is selfcomplementary
– Bases can pair up to
form a hairpin as seen
in the lower panel
6-48
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
6-49
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
6-50
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
6-51
Rho Affects Chain Elongation
• There is little effect of r on transcription
initiation, if anything it is increased
• The effect of r on total RNA synthesis is a
significant decrease
• This is consistent with action of r to
terminate transcription forcing timeconsuming reinitiation
6-52
Rho Causes Production of
Shorter Transcripts
• Synthesis of much smaller RNAs occurs in
the presence of r compared to those
made in the absence
• To ensure that this due to r, not to RNase
activity of r, RNA was transcribed without
r and then incubated in the presence of r
• There was no loss of transcript size, so no
RNase activity in r
6-53
Rho Releases Transcripts from
the DNA Template
• Compare the sedimentation of transcripts
made in presence and absence of r
– Without r, transcripts cosedimented with the
DNA template – they hadn’t been released
– With r present in the incubation, transcripts
sedimented more slowly – they were not
associated with the DNA template
• It appears that r serves to release the
RNA transcripts from the DNA template
6-54
Mechanism of Rho
• No string of T’s in the rdependent terminator,
just inverted repeat to
hairpin
• Binding to the growing
transcript, r 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
6-55