Transcript Chapter 06 Lecture PowerPoint - McGraw Hill Higher Education

Lecture PowerPoint to accompany
Molecular Biology
Fifth 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
Summary
• The key player in the transcription process is
RNA polymerase
• The E. coli enzyme is composed of a core,
which contains the basic transcription
machinery, and a s-factor, which directs the core
to transcribe specific genes
6-4
6.2 Promoters
• Why was the core RNA polymerase
capable of transcribing nicked DNA in the
previous table?
• Nicks and gaps are good sites for RNA
polymerase to bind nonspecifically
• The presence of the s-subunit permits
recognition of authentic RNA polymerase
binding sites 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 the temperature is
lowered, the binding
of RNA polymerase to
DNA decreases
dramatically
• Higher temperatures
promote DNA melting
and encourage RNA
polymerase binding
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 a promoter
• 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
Summary
• The s-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 promoter complex
and is stimulated by s
• The s-factor can therefore select which genes
will be transcribed
6-10
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 (or most
common sequence) for each of these boxes
6-11
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
6-12
UP Element
• The UP element is upstream of the core
promoter, stimulating transcription by a
factor of 30
• UP is associated with 3 “Fis” sites which
are binding sites for the transcriptionactivator protein Fis, not for the
polymerase itself
6-13
The rrnB P1 Promoter
• Transcription from the rrn promoters
respond
–Positively to increased concentration of iNTP
–Negatively to the alarmone ppGpp
6-14
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
6-15
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
6-16
Sigma Stimulates Initiation of Transcription
• In this first experiment
stimulation by s appears
to cause both initiation
and elongation
• Or stimulating initiation
by s provides more
initiated chains for core
polymerase to elongate
• Further experiments by
the same group proved
that s does not stimulate
elongation
6-17
Reuse of s
• During initiation s can be recycled for additional
use with a new core polymerase
• The core enzyme can release s which is then
free to associate with another core enzyme
6-18
Fluorescence Resonance Energy Transfer
• The s-factor changes its relationship to the core
polymerase during elongation
• It may not dissociate from the core but actually shift
position and become more loosely bound to core
• To answer this question Fluorescence Resonance
Energy Transfer (FRET) was used as it relies on
two fluorescent molecules that are close enough
together to engage in transfer of resonance energy
• FRET allows the position of s relative to a site on
the DNA to be measured without using separation
techniques that might displace s from the core
enzyme
6-19
FRET Assay for s Movement Relative to DNA
6-20
Models for the s-Cycle
• The obligate release version of the s-cycle
model arose from experiments performed by
Travers and Burgess that proposed the
dissociation of s from core as polymerase
undergoes promoter clearance and switches
from initiation to elongation mode
• The stochastic release model proposes that s
is indeed released from the core polymerase but
that there is no discrete point of release during
transcription and that the release occurs at
random - a preponderance of evidence favors
this model
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
Experiment to locate the region of early
promoter melted by RNA Polymerase
6-23
Promoter Clearance
• RNA polymerases have evolved to
recognize and bind strongly to promoters
• This poses a challenge when it comes
time for promoter clearance as those
strong bonds must be broken in order for
polymerase to leave the promoter and
enter the elongation phase
6-24
Promoter Clearance
• Several hypotheses have been proposed
• The polymerase cannot move enough
downstream to make a 10-nt transcript without
doing one of three things:
- transient excursion: moving briefly
downstream and then snapping back to the
starting position
- inchworming: stretching itself by leaving its
trailing edge in place while moving ots leading
edge downstream
- scrunching: compressing the DNA without
moving itself
6-25
Abortive Transcription, Scrunching and
Promoter Clearance
• Ebert and colleagues performed several
experiments to distinguish between the hypotheses
• Using E.coli polymerase the authors concluded
that approximately 100% of all transcription cycles
involved scrunching, which suggested that
scrunching is required for promoter clearance
• The E.coli polymerase achieves abortive
transcription by scrunching: drawing downstream
DNA into the polymerase without actually moving
and losing its grip on promoter DNA
• The scrunched DNA could store enough energy to
allow the polymerase to break its bonds to the
6-26
promoter and begin productive transcription
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
6-27
Homologous Regions in Bacterial s Factors
6-28
E. coli s70
• Four regions of high sequence similarity
are indicated
• Specific areas that recognize the core
promoter elements are the -10 box and –
35 box
6-29
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
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
6-30
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
6-31
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
6-32
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
6-33
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 (CTD)
• Results in very strong
interaction between
polymerase and promoter
• This produces a high level of
transcription
6-34
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
6-35
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
6-36
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 the
nucleotide-binding site during the
initiation phase
6-37
Structure of the Elongation Complex
• This section will examine how well
predictions have been borne out by
structural studies
• 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?
6-38
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
6-39
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
6-40
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
6-41
Structure of the Elongation Complex
• The X-ray crystal structure of the Thermus
thermophilus RNA polymerase elongation
complex in 2007 revealed several important
observations
– a valine residue in the E’ subunit inserts into
the minor groove of the downstream DNA
– the downstream DNA is double-stranded up to
and including the +2 bse pair
– the enzyme can accommodate nine base
pairs of RNA-DNA hybrid
– the RNA product in the exit channel is twisted
into the shape it would assume as 1/2 of an Aform dsRNA
6-42
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 rewind behind it
• Strain introduced into the template DNA ahead
of the transcription bubble is relaxed by
topoisomerases
6-43
Pausing and Proofreading
• RNA polymerase frequently pauses, or even
backtracks, during elongation
• Pausing allows ribosomes to keep pace with the
RNA polymerase, and it is the first step in
termination
• Backtracking aids proofreading by extruding the
3’-end of the RNA out of the polymerase, where
misincorporated nucleotides can be removed by
an inherent nuclease activity of the polymerase,
stimulated by auxiliary factors
6-44
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
rho (r, these are rho or r-dependent
terminators
6-45
Rho-Independent Termination
• Intrinsic or rho-independent termination
depends on terminators of 2 elements:
– Inverted repeats followed immediately by
– T-rich region in the nontemplate strand of the
gene
• An inverted repeat predisposes a
transcript to form a hairpin structure due to
complementary base pairing between the
inverted repeat sequences
6-46
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
6-47
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
6-48
Model of Intrinsic Termination
Bacterial terminators act by:
• Base-pairing of something to
the transcript to destabilize
RNA-DNA hybrid
– Causes hairpin to form
• This causes transcription to
pause
– a string of U’s incorporated
just downstream of hairpin to
destabilize the hybrid and the
RNA falls off the DNA template
6-49
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
6-50
Rho Affects Chain Elongation
• There is little effect of rho or r on
transcription initiation, if anything it is
increased
• The effect of rho or r on total RNA
synthesis is a significant decrease
• This is consistent with action of rho or r to
terminate transcription forcing timeconsuming reinitiation
6-51
Rho Causes Production of Shorter
Transcripts
• Synthesis of much smaller RNAs occurs in
the presence of rho or r compared to
those made in the absence
• To ensure that this due to r itself and 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
6-52
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
6-53
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
6-54
Summary
• Using the trp attenuator as a model rho-independet
terminator revealed two important features:
1 - an inverted repeat that allows a hairpin to for at the
end of the transcript
2 - a string of T’s in the nontemplate strand that results
in a string of weak rU-dA base pairs holding the
transcript to the template strand
• Rho-dependent terminators consist of an inverted
repeat, which can cause a hairpin to form in the
transcript but no string of T’s
6-55