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Molecular Biology (3/30~4/25, 2007)
Ch. 9
What is transcription?
How transcription works?
Stages
Machinery
Molecular mechanism
Ch. 10,11
How transcription is regulated?
Regulators
Mechanisms
Ch. 12, 11
Examples of transcriptional regulation
Phage strategy
RNA silencing
沈湯龍
(Tang-Long Shen) 助理教授
細胞生物學
一號館315室
Tel: 3366-4998; E-mail: [email protected]
Transcription in Prokaryotes vs. Eukaryotes
Prokaryotic Cell
Because there is no nucleus to separate the processes of
transcription and translation, when bacterial genes are
transcribed, their transcripts can immediately be
translated.
Eukaryotic Cell
Transcription and translation are spatially and
temporally separated in eukaryotic cells; that is,
transcription occurs in the nucleus to produce a premRNA molecule.
The pre-mRNA is typically processed to produce the
mature mRNA, which exits the nucleus and is translated
in the cytoplasm.
Transcription in prokaryotes
The basis of life
What is transcription?
Central Dogma of Biology:
DNA → RNA → protein
☆ Gene Expression: Transcription
Transcription = DNA → RNA
☆ Gene functions (majority) are expressed as the proteins they encode: Translation
Translation = RNA → protein
RNA is structurally similar to DNA
But……..
Gene Transcription: DNA → RNA
genetic information flows from DNA to RNA
by RNA polymerase
RNA is identical in sequence with one strand of the DNA (but
T→U), called coding strand.
Four stages of transcription:
1. Promoter recognition and initial melting
(binary complex formation)
2. Initiation (ternary complex formation)
3. Elongation
4. Termination
Transcription Unit
RNA polymerase
Transcription
unit
binding
A transcription unit is the
distance between sites of
initiation and termination by
RNA polymerase; may include
more than one gene (particularly
in prokaryotes).
release
May include more than
,
one gene
5’
no number 0
5’
3’
(Primary transcript)
3’
A relative location
on a linear sequence
mRNA
How transcription works?
Basic principles of transcription
Template recognition: polymerase and duplex
DNA
Initiation: polymerase* and promoters
Elongation: RNA polymerase
Termination: terminator
abortive
initiation
Initiation
• Binding of an RNA polymerase to the
dsDNA
• (Slide) to find the promoter
• Unwind the DNA helix
• Synthesis of the RNA strand at the start
site (initiation site), this position called
position +1
Transcription Bubble
To fulfill the principle process of transcription, that is
complementary base pairing, a transient bubble has to be created.
Two strands of DNA are separated
(about 12~14 bp in length).
Template strand is used to synthesize
a complementary sequence of RNA.
The length of RNA-DNA hybrid
within the bubble is about 8~9 bp.
As RNA polymerase moves along the
DNA, the transient bubble moves
along with and the RNA chain grows
continuously.
Transcription Bubble
RNA-DNA hybrid length
Ternary Complex:
~ 8 to 9 bases, it is short and
transient
Polymerase-DNA-RNA
Function of RNA Polymerase
Unwinding and Rewind DNA
NTPs polymerized to a RNA chain
Moving in the DNA
About 25-base RNA molecule associated with
the ternary complex at any moment.
Elongation
5’
RNA extension
3’
Progression of
transcription bubble is
association with
RNA polymerase
movement on DNA
Movement models
1. Sliding:
2. inchworm
DNA rewind behind
DNA unwind ahead
RNA
Reaction in Transcription (RNA polymerization)
DNA replication
RNA polymerization
Direction 5’ to 3’
~40 nt/sec
5
4
3
NTP
γ
2
β
Substrates
ATP, UTP, GTP, CTP
5’ → 3’ ~800 bp/sec
α
NTP
5
4
Phosphate
α,β,γ
N → C termini ~15 aa/sec
3
2 1
NTP
5
Nucleotide
γ
3
β
Ribose
5C -- 1,2,3,4, 5
α
NTP
5
3
Protein translation
Stages of transcription
(5’) Promoter
: closed complex
Terminator (3’)
: open complex
Binary
Promoter clears
Bubble moves on
Ternary
Abortive initiation: to ensure the initiation in a right way.
Movement models
1. Sliding: common
2. inchworm
(before the 10th base is added on nascent RNA chain within the bubble)
move
Extending RNA chain is accomplished with RNA poly (bubble)
moves along DNA.
The bases after 9th enable added on the growing RNA chain.
Recognize termination signal
Release RNA chain (by disrupt RNA:DNA hybrid)
Dissociation of RNA pol
Machinery in transcription
Transcription in Prokaryotes
RNA polymerase
Prokaryotes have a single RNA polymerase
enzyme--synthesizes mRNAs, rRNAs, and tRNAs
Transcribe over > 1000 transcription units. The complexity is modified by
interacting with diverse regulatory factors.
Eukaryotes have three RNA polymerase Enzymes:
RNA Pol I
rRNA
RNA Pol II
mRNA
RNA Pol III
tRNA, 5S rRNA
E. coli RNA polymerase
RNA polymerase binds to the promoter
Core enzyme + sigma factor = holoenzyme
155 KD
36.5 KD
11 KD
36.5 KD
70 KD
Initiation only
151 KD
465kD
Both initiation & elongation
Structure and functions of E. coli RNA Polymerase
2 a subunits
Enzyme assembly, Promoter
recognition, factor binding
b subunit
Catalytic Center
b' subunit
Catalytic Center
Template-binding
s subunit
Promoter specificity
Eubacteria RNA polymerase (Pol)
About 7000 RNA polymerase molecules are
present in an E. coli cell.
Most of them are engaged in transcription.
In a short period of time, 2000-5000 Pol molecules
can be synthesized.
E. coli Polymerase: α subunit
•
•
•
•
Two identical subunits in the core enzyme
Encoded by the rpoA gene
Required for core protein assembly
May play a role in promoter recognition and
regulatory factors interaction
• ADP-ribosylation on an arginine upon T4
infection
E. coli polymerase: b subunit
1.
2.
•
Encoded by rpoB gene.
The catalytic center of the RNA polymerase
Rifampicin (used for anti-tuberculosis): bind to the β
subunit (12A away from active site), and inhibit
transcription initiation. Blocking the path for extending
RNA chain beyond 2-3 nts. Mutation in rpoB gene can
result in rifampicin resistance.
•
Streptolydigins：resistant mutations are mapped to
rpoB gene as well. Inhibits transcription elongation but
not initiation.
3. b subunit may contain two domains responsible for
transcription initiation and elongation
E. coli polymerase: b’ subunit
1.
2.
•
3.
Encoded by the rpoC gene .
Binds two Zn 2+ /Mg 2+ ions and may participate in the
catalytic function of the polymerase
Heparin:binds to the b’ subunit and inhibits
transcription in vitro due to it competes with DNA for
binding to the polymerase.
b’ subunit may be responsible for binding to the
template DNA .
E. coli polymerase: s factor
1.
2.
3.
4.
5.
Many prokaryotes contain multiple s factors to
recognize different promoters. The most common s
factor in E. coli is s70. (differential specificity)
Binding of the s factor converts the core RNA pol
into the holoenzyme.
s factor is critical in promoter recognition, by
decreasing the affinity of the core enzyme for nonspecific DNA sites (104) and increasing the affinity for
the corresponding promoter
s factor is released from the RNA pol after initiation
(RNA chain is 8-9 nt)
Less amount of s factor is required in cells than that
of the other subunits of the RNA pol.
Holoenzyme on promoter recognition
(Core enzyme + sigma factor = holoenzyme)
Core enzyme has the ability to synthesize RNA on a DNA template,
but cannot initiate transcription at the proper sites.
Holoenzyme has ~104-fold lower affinity for loose binding
complexes than core. About 60 min half-life reduce to <1 sec.
Holoenzyme has ~103-fold higher affinity for specific binding
to promoters than core with a half life of several hours.
Totally, sigma factor can result in
107 increase in DNA binding specificity.
Core enzyme does not distinguish between
promoters and other sequences of DNA.
Sigma factor is required only for initiation
reversible
Wide range
Faster
Tight binding
Fastest
Less than 10 bases
Beyond 10 bases leads to elongation
Slow
Recycle of sigma factor for the utilization of core enzyme
Sigma factor is much less in number than core enzyme
Evidence:
1/3 of sigma factors are not associated with
core enzyme while elongation
recycled
Immediately after initiation
Molecular structure of RNA polymerases
in functioning
Architecture of RNA polymerases (prokaryotes)
T7 RNA polymerase (<100 kD)
Bacterial RNA polymerase (465kD)
Multiple subunits:
2α+β+β’+(σ)
25A wide
Enzyme movement
~200 nts/sec
Specificity recognition between enzyme and
DNA bases (upstream of startpoint +1)
~40nts/sec
A channel/groove on the surface ~25A wide
forms a path for DNA.
Path holds for 16 bp in prokaryotes
25 bp in eukaryotes
More DNA bp can reside on the enzyme
Further crystal structure will provide more direct and detailed view in a molecular level.
Architecture of RNA polymerases (eukaryotes)
Yeast RNA polymerase contains 12 subunits (10 are shown here)
Nevertheless, it shares similar organization as bacterial one.
Cleft between two
large subunits forms
as an active center
A channel/groove on the surface
forms a path for DNA.
25 bp DNA can be held in the path.
Ternary Complex
Channel within RNA polymerase
Active center
+
Enzyme
movement
DNA in and out
DNA out
rudder
RNA dissociated
DNA turns
DNA in
Flexible ss DNA
RNA flipped out
Rigid straight duplex DNA
entry
(control by bridge protein)
How many bp(s) in the bubble?
Contact among the ternary structure in the active site
These contacts can stabilize the single strand nucleic acid chains.
Cycle of making and breaking bonds between enzyme and nucleic acids
nt enters, adds,
and interacts with
the bridge protein
nt still interacts with the
bridge protein, which
leads the protein to
bending due to Pol
moves one bp forward.
Meanwhile, bridge blocks
free nt enters.
Finally, bridge releases
Its interaction with newly
added nt on RNA chain.
Change in conformation of “bridge” protein is closely related
to translocation of the enzyme along the nucleic acid.
How does RNA polymerase find promoter sequences?
Random diffusion
RNA polymerase found promoters is very faster.
Diffusion in the whole genome cannot support
this fast.
Directed walk
(Direct displacement)
vs.
Random walk
No DNA protein is known to work in this way
Enzyme moves preferentially from a weak site to a strong site
Transitions in shape and size of RNA polymerase during transcription
Covered DNA length
75-80 bp
(-55 to +20)
60 bp
(-35 to +20s)
30-40 bp
(interact w/ RNA pol)
How to resume the stalled/pausing RNA polymerase?
Cleavage 3’ end of RNA chain
(Create a 3’-OH for further polymerization)
Backtracks of RNA polymerase as a whole
A constant distance between active site and frond end
To correct mispositioned template during stall
Accessory factors are needed such as:
GreA and GreB for E. coli RNA polymerase
TFIIS for eukaryotic RNA polymerase II
One more function of RNA polymerase:
* cleavage activity is from RNA polymerase itself.
unwind
Rewind
DNA/RNA binding
polymerize RNA
Sequence elements in Transcription
Promoter
Coding sequence
Terminator
What is a promoter?
•The sequence of DNA needed for RNA polymerase to bind to
the template and accomplish the initiation reaction.
•Its structure (not transcribed) is the signal (others are
needed to be converted into RNAs or proteins).
It is a cis-acting site.
•Different from sequences whose role is to be transcribed or
translated.
What signal (structure) of a promoter provides?
AT has only 2 H-bonds, which is easier to be broken
(Open binary complex formation)
(recognition domain
(Closed binary complex formation)
(i.e. the distance of separation between -10 and -35;
intermediate sequence is irrelevant)
Pribnow, D.: Nucleotide Sequence of an RNA Polymerase Binding Site at an Early T7 Promoter. PNAS 72, 784 (1975).
Pribnow, D.: Bacteriophage T7 early promoters: nucleotide sequences of two RNA polymerase binding sites. J. Mol. Biol. 99, 419 (1975).
Schaller, H. et al.: Nucleotide Sequence of an RNA Polymerase Binding Site from the DNA of Bacteriophage fd. PNAS 72, 737 (1975).
The sequence comparison of five E. coli promoters
Consensus TTGACA TATAAT
Consensus:
the most common base sequence to appear at such points on the DNA helix;
there may be variations in various organisms
Prokaryotic promoters display four conserved features:
1. Startpoint: >90% PURINE (A or G)
2. -10 consensus sequence (Pribnow box)--TAtAaT
T80 A95 t45 A60 a50 T96
3. -35 consensus sequence--TTGACa
T82 T84 G78 A65 C54 a45
4. Distance (spacing) between the -10 and -35 sequences
(The distance is critical in holding the two sites at the appropriate separation for
the geometry of RNA polymerase.)
5. UP element. TA rich sequence upstream of promoter.
Functions of promoter domains
-35 recognition domain
Closed binary complex formation
-10 unwinding domain: due to A-T pairs
need lower energy to disrupt (melt)
Open binary complex formation
Sequence around the startpoint (+1 to +30):
influences the initiation event.
Rate of promoter clearance
Other ancillary proteins may help RNA polymerase to recognize deficient promoters.
Other structures may exist in a promoter
A-T rich sequence
It interacts with the α subunit of the RNA polymerase,
which to ensure the higher gene expression.
100-fold
variation
in vitro
Down mutation: mutations are tend to be concentrated in the most highly conserved positions.
Up mutation: less cases happen within promoters
RNA polymerase-promoter interactions
A promoter with consensus sequences for the -10 and -35 regions (boxed) is shown;
the sequences of actual promoters deviate from those shown here.
The "jaws" of RNA polymerase are shown on the right of the molecule. This region of
the RNA polymerase would grasp the DNA downstream of the catalytic site. Contacts
between RNA polymerase and promoter DNA are shown by the solid lines. Not all
contacts occur in every RNA polymerase-promoter interaction, but in all known cases
(including promoters activated by regulator proteins), at a minimum, some contacts
between and the 10 region appear to be required.
J Bacteriol, June 1998, p. 3019-3025, Vol. 180, No. 12
-10 sequence (Pribnow box)
• 6 bp sequence which is centered at around the –10
position (Pribnow, 1975).
• A consensus sequence of TATAAT
• The first two bases(TA) and the final T are most highly
conserved among other E. coli promoters
• This hexamer is separated by 5 to 9 bp from position
+1, and the distance is critical
• DNA unwinding is initiated at promoter by the
polymerase
-35 sequence:
enhances recognition and interaction with the
polymerase s factor
• A conserved hexamer sequence around position
–35
• A consensus sequence of TTGACA
• The first three positions (TTG) are the most
conserved among E. coli promoters.
• Separated by 16-18 bp from the –10 box in 90% of
all promoters
Transcription start site
The sequence around the start site influences initiation
• A purine (A or G) in 90% of all genes
• Often, there are C and T bases on either side of the start
site nucleotide (i.e. CGT or CAT)
Promoter efficiency (1)
• There is considerable variation in sequence
between different promoters, and the
transcription efficiency can vary by up to 1000fold .
• The –35 sequence, -10 sequence, and
sequence around the start sites all influence
initiation efficiency.
Promoter efficiency (2)
• The sequence of the first 30 bases to be
transcribed controls the rate at which the RNA
polymerase clears the promoter, hence
influences the rate of the transcription and the
overall promoter strength .
• Strand separation in the initiation reaction
• Some promoter sequence are not strong
enough to initiate transcription under normal
condition, activating factor is required for
initiation. For example, Lac promoter Plac
requires cAMP receptor protein (CRP )
DNA unwinding
• Necessary to unwind the DNA so that the
antisense strand to become accessible for
base pairing, carried out by the polymerase.
• Negative supercoiling enhances the
transcription of many genes but not all (e.g.
gyrase) by facilitating unwinding .
• The initial unwinding of the DNA results in
formation of an open complex with the
polymerase and this process is referred to as
tight binding
Supercoiling during transcription
At initiation
∵ Supercoiled structure requires less free energy for the initial melting of DNA
∴ it enhances the efficiency of transcription in vitro
After initiation DNA is rotated during RNA pol movement; front is overwound and behind is released.
A twin domain on transcribing DNA formed
RNA polymerase binds to one face of DNA
(-9 to +3 for unwinding)
Touch down
extension
recognition
contact
initiation
Sigma factor controls promoter recognition
Different sigma is used for distinct responses
"housekeeping"
starvation/stationary
extracytoplasmic stress
nitrogen-limitation
flagellar sigma factor
The specificity is determined by recognizing different
consensus sequences in promoters
Bacillus subtilis sigma factors
s factor
gene
function
43
rpoD, sigA
housekeeping
28
sigD
flagella/chemotaxis
29
spoIIGB, sigE sporulation
30
spo0H, sigH
sporulation
32
sigC
?
37
sigB
?
spoIIAC spoIIAC
sporulation
?
spoIIIC
sporulation
gp28
SPO1 28
phage middle
gp33/34 SPO1 33,34
phage late
Sigma factors may be organized into cascades
A new sigma factor displaces the previous sigma factor
Sigma factors directly contact DNA
which contributes the binding specificities of sigma factors
(most conserved)
melting
2.3
2.4
4.2
domain1
15A
Release N-terminal
autoinhibition due
conformation change
via interaction with
RNA polymerase
conformation change
20A
Free Holo: inside the active site
Complex: displace from active site
Coding stand
Transcription initiation
DNA-dependent RNA polymerases are promoter binding,
DNA strand melting,
RNA chain initiation and
nascent RNA chain formation, and
abortive RNA synthesis occurs
finally escape from the promoter sequences.
rate-limiting for the synthesis of productive RNAs
What is the role of sigma factor in abortive initiation/promoter clearance (escape)?
Elongation
Elongation
• Add ribonucleotides to the 3’-end (OH
group)
• The RNA polymerase extend the growing
RNA chain in the direction of 5’ 3’ （E.
coli: 40 nt/sec)
• The enzyme itself moves in 3’ to 5’ along
the antisense DNA strand.
RNA chain elongation
• σFactor is released to form a ternary complex
of the pol-DNA-RNA (newly synthesized),
causing the polymerase to progress along
the DNA (promoter clearance)
• Transcription bubble (unwound DNA region,
~ 17 bp) moves along the DNA with RNA
polymerase which unwinds DNA at the front
and rewinds it at the rear
• 3’ part of RNA forms hybrid helix (ca. 12bp)
with antisense DNA strand.
• The E. coli polymerase moves at an average
rate of ~ 40 nt per sec, depending on the
local DNA sequence.
Termination
• The dissociation of the transcription
complex from the template strand and
separation of RNA strand from DNA
• Occurring at the terminator (often
stem-loop or hairpin structure), some
need rho protein as accessory factor.
it is a regulatory event
Hence, it is possible to readthrough the terminator (anti-termination)
in a signal-dependent manner.
RNA chain termination
• Termination: dissociation of RNA > re-annealing of DNA > release of
RNA pol
• Terminator sequence (stop signal):
• RNA hairpin very common
• Accessory rho protein
The DNA sequences required for termination are
located prior to the terminator sequence.
Formation of a hairpin in the RNA may be necessary
RNA hairpin structure: an intrinsic terminator
near the base of the stem.
Hairpin leads RNA pol to slow/pause
The rU.dA RNA –DNA hybrid has an unusually weak base-paired structure;
it requires the least energy of any RNA-DNA hybrid to break the association
between the two strands.
A model for intrinsic termination
Rho-dependant termination
• Some genes contain terminator
sequences requiring an accessory
factor,the rho protein (ρ) to mediated
transcription termination
• Rho binds to specific sites in the
single-stranded RNA
• Rho hydrolyses ATP and moves
along the nascent RNA towards the
transcription complex then enables
the polymerase to terminate
transcription
Termination efficiency determinants:
@ The Sequence of the hairpin
@ The length of the U-run
@ Sequences both upstream and downstream of the intrinsic terminator
@ Ancillary proteins
@ others
rich
poor
A bias sequence preceding actual terminator site (RNA) is important for termination efficiency
(rho dependent terminator).
~275kD