Transcription

Vipin
Shankar
The Central Dogma
 The information on the DNA has to
be converted to the protein
quotient of the cell.
 This is carried in a two step
process.
 DNA is first transcribed to RNA.
 The RNA is then translated to
proteins.
Transcription
 Chemically and enzymatically
similar to DNA replication.
 Both involve the synthesis of a new
strand of nucleotide
complimentary to a DNA template
strand.
 The most notable difference
between the 2 is that, in
transcription, the new strand is a
RNA.
Transcription v/s Replication
 RNA Polymerase: the enzyme that
catalyze the reaction, does not
require a primer.
 The RNA product does not remain
base paired to the template DNA.
 Transcription is less accurate than
replication. (error rate of 1 in 10,000
compared to 1 in 10,000,000)
Transcription v/s Replication…
 Transcription selectively copies
only certain parts of the genome
and makes anything from one to
several hundred (or even thousand)
copies of any given section.
RNA Polymerases
 Essentially performs the same reaction
in all cells from bacteria to humans.
 All RNA Polymerases, share some
common features.
 The cellular RNA Pol is a multi-subunit
complex protein.
 Bacteria has a single RNA Pol,
Eukaryotic cells have 3; RNA Pol I, II &
III.
RNA Polymerases…
 RNA Pol-II carries out transcription
of mRNAs in eukaryotes.
 RNA Pol-II & III are involved in
transcribing specialized RNA –
encoding genes.
RNA Polymerases…
 The bacterial core enzyme is
composed of 2 copies of α
subunits, one each of β, β’ & ω
subunits.
 The shape of each enzyme complex
resembles a crab claw.
 The two pincers of the crab claw
are made up of the two large
subunits β & β’.
RNA Polymerases…
 The active site is at the base of the
two pincers called the active cleft.
 The active cleft binds 2 Mg2+ ions.
 There are various channels that
allow DNA, RNA and ribonuclotides
into and out of the enzymes’ active
cleft.
Steps in transcription
 3 phases are identified.
 Initiation
 Elongation
 Termination
Initiation
 A region called the Promoter on the
DNA binds to RNA Pol.
 The promoter-polymerase complex
undergoes structural changes.
 The DNA around the point where
transcription starts unwinds
producing a Transcription Bubble.
Initiation…
 Transcription always occurs in 5’ to 3’
direction.
 Only one of the DNA strand act as a
template.
 The RNA Pol binds to the promoter in a
defined orientation, and the same
strand is transcribed.
 The choice of the promoter determines
which strand is to be transcribed and
also the rate of transcription.
Initiation…
 Involves 3 steps.
 Initial binding of RNA Pol to a promoter to
form a closed complex.
 The closed complex undergoes transition to
the open complex in which the DNA strand
separates for over 14bp.
 The unwinding of the double helix, initiates
transcription.
 The first two rNTPs are brought to the active
cleft and the system begins polymerization.
Initiation…
 The enzyme then moves along the template
 Opening the double helix ahead of the site of
polymerization.
 Incorporating the complimentary rNTP.
 Re-annealing the double helix behind the
polymerization site.
 The incorporation of the first 10 rNTPs is
inefficient.
 Once an enzyme gets further than 10bp, it
is said to have escaped the promoter.
 At this point it has formed the Stable
Ternary Complex.
Elongation
 Once the RNA Pol has synthesized a
short segment of RNA, it enters the
elongation phase.
 This requires a conformational change
in RNA Pol.
 During elongation the enzyme performs
 Unwinds DNA in front & re-anneals it
behind.
 Dissociates the RNA chain from the
template as it moves along.
 Performs proof reading function.
Termination
 Once the polymerase has
transcribed the length of the gene
it stops and releases the RNA
product.
 In some cells there are well
characterized termination
sequences that trigger termination.
Transcription in Bacteria
 An additional protein called the σ-
factor.
 The form of RNA Pol with the σ-
factor is called the holoenzyme.
 In the case of E. coli, the
predominant σ-factor is called σ70.
 σ70 recognizes the promoter.
Transcription in bacteria…
 The promoter region is a stretch of
2 conserved sequences at the -10
and -35 region.
 The consensus sequence can be
derived by comparing the promoter
regions of various organisms.
 Promoters with sequences closer to
the consensus sequence are strong
promoters.
Transcription in bacteria…
 An additional element that binds to
RNA Pol is found is some strong
promoters – UP-element.
σ-factor mediates RNA Pol
binding on the promoter
 The σ70 has a helix-turn-helix
motif.
 One of the helices interacts with
the -35 region.
 The -10 region is recognized by
another helix.
 DNA melting is initiated within the
-10 region.
σ-factor mediates RNA Pol
binding on the promoter…
 The UP-element is not recognized
by σ70.
 This is recognized by the αCTD (c
terminal of α subunit).
Transition to the open
complex
 The transition involves
 Unwinding of the double helix
between -11 and +3 regions.
 Conformational change in the
protein.
 In case of the σ70 bearing
holoenzyme, this is called
isomerization and does not require
energy.
Transition to the open
complex…
 This happens due to spontaneous
conformational change.
 Isomerization is irreversible.
 Completion of isomerization
guarantees initiation of
transcription.
RNA Pol does not require a
primer
 The initiating rNTP is brought into the
active cleft and is held stably on the
template.
 The next NTP is then presented with the
correct geometry and polymerization is
carried out.
 The enzyme makes specific interactions
with the initiating rNTP.
 Under most conditions a rATP is used
as the initiating rNTP.
Abortive initiation
 Once RNA synthesis begins, RNA Pol
initially synthesizes short RNA (<10nt in
length).
 Instead of being elongated these are
released and RNA Pol restarts with a
new RNA.
 This is probably because of the
positioning of the σ-factor, which
hinders the RNA exit channel.
 The σ-factor later dissociates, and
elongation begins.
The elongating polymerase is
a processive machine
 During elongation
 The double helix DNA enters the enzyme between
the pincers.
 At the opening of the catalytic cleft the double helix
is unwound, the strands separate and follow
different paths and are later rejoined behind the
elongating polymerase.
 The rNTPs enter through a channel and are added to
the growing RNA.
 Only 8 or 9 NTPs of the growing RNA remain base
paired to the template.
 The remainder of the RNA is pealed off and are
directed out through the RNA exit channel.
..\Animations\Transcription.MOV
Proofreading
 2 methods.
 Pyrophosphorolytic editing:
 A simple back reaction.
 Removes an incorrectly inserted rNTP
and reincorporates the correct one.
 Hydrolytic editing:
 Backtracks one or more NTPs and
cleaves the RNA product, removing the
error containing sequence.
 Stimulated by Gre factors.
Termination in bacteria
 Sequences called terminators
trigger termination
 2 different modes
 Rho-independent
 Rho-dependent
Rho-independent termination
 Also called intrinsic termination.
 The terminator sequence
comprises of an inverted repeat.
 This is followed by a a stretch of
about 8 A:T base pairs.
 These elements do not affect RNA
Pol until after they are transcribed.
 These elements function in the
RNA rather than in the DNA
Rho-independent
termination…
 When RNA Pol transcribes an
inverted repeat, the resulting RNA
forms a hairpin structure.
 The hairpin causes termination by
disrupting the elongation complex.
Rho-dependent termination
 Less well characterized terminator
sequences.
 Requires the action of ρ-protein.
 This is a ring shaped protein with 6
identical subunits.
 Binds to the single stranded RNA at
sites called rut (Rho utilization sites).
 ρ-protein has ATPase activity.
 Once attached to the transcript, it uses
the energy derived from ATP to wrest
the RNA from the polymerase complex.
Transcription in eukaryotes
 Eukaryotes have 3 different
polymerases.
 Several initiation factors are required
General transcription Factors (GTFs).
 Additional factors
 Mediator complex
 DNA binding regulatory proteins

 Chromatin modifying enzymes

Are also required.

Types of RNA polymerases and their function

Types of Transcription factors

Promoters
 The core promoter refers to the minimal
sequence elements required for accurate
transcription initiation by Pol II.
 Typically 40 nts long, extending either
upstream or downstream.
 4 elements found in ek promoters are
 TFIIB recognition element.
 The TATA Box.
 The Initiator (Inr).
 The downstream promoter element (DPE).
Regulatory elements and factors recognizing them
RNA Pol II forms a pre-
initiation complex
 TFIID binds to the TATA box via the TBP.
 This association distorts the -10 region and
slightly unwinds the DNA double helix.
 The TBP-DNA complex recruits TFIIA, TFIIB,
TFIIF together with polymerase.
 The polymerase is in complex with TFIIE,
TFIIH and the mediator complexes which bind
upstream to the TATA box.
 This assembly of proteins on the DNA double
helix is called the pre-initiation complex.
The pre-initiation complex…
 The DNA melting is mediated by TFIIH.
 Action of TFIIH is similar to Helicase
and requires energy.
 Once the pre-initiation complex is
formed the polymerase begins to add
rNTPs.
 The system first enters an abortive
initiation phase similar to that in
prokaryotes.
 The phosphorylation of RNA Pol-II CTD
results in escape from the promoter and
the triggering of elongation.
Elongation
 Elongation requires specific factors
called elongation factors.
 These factors are of 2 types
 Those that stimulate elongation.
 Those that bring about RNA editing.
 Both these factors are attached to the
RNA Pol-II CTD.
 There is an overlap of elongation and
RNA editing.
 RNA editing is simultaneous with
elongation.
Termination
 Termination is less understood in
eukaryotes.
 It is proposed that the 3 polymerases
use different mechanisms for
termination.
 Pol-I uses a mechanism similar to Rho-
dependent termination.
 Pol-III terminates after transcribing a
termination sequence which contains a
string of Us.

..\Animations\Transcription and translation in ek.MOV