molecular biology transcription mb 07
TRANSCRIPT
MOLECULAR BIOLOGY
TRANSCRIPTION
MACHINERY
Dr. Aga Syed SameerCISR Lecturer (Demonstrator)
Department of Biochemistry,
Medical College,
Sher-I-Kashmir Institute of Medical Sciences,
Bemina, Srinagar, Kashmir, 190018. India.
PROMOTERS
PROMOTERS
In bacteria, the specific initiation sites for RNA
polymerase attachment are called as promoters
They are located usually upstream from the
sequences/gene to be transcribed
The transcription start is generally assigned a
position +1 and the promoter sequences are
assigned a negative number reflecting the
distance upstream from the start site
E. coli promoter consists of two segments both of
which contain highly conserved hexameric
sequence
PROMOTERS
-10 box: Also referred as Pribnow box has a
consensus sequence of TATAAT of which first two
& last one are highly conserved. This is separated
by 5-8bp intervening sequence from the start
site whose distance is critical
-35 box: It has a consensus sequence of TTGACA, of
which first three are highly conserved. This is
separated by 16-18bp from the -10 box
In about 90% of the genes transcription start
site is a purine and often it remains flanked on
either side by C & T bases (CGT or CAT)
INITIATION & ELONGATION
The process of initiation involves the recognition
of the transcription start site
In prokaryotes it is done by the RNA polymerase
itself with the help of σ factor of the
holoenzyme
σ factor recognizes -35 box and binds to it forming
a closed promoter complex and then facilitates
the opening up of the helix between the
polymerase and the non-template strand
After the successful initiation it dissociates from
the enzyme so that elongation phase is paved
But in eukaryotes, initiation is facilitated by about
30 or more proteins called as general
transcription factors (GTFs)
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PROMOTER CLEARANCE
The carboxyl end of RNA Polymerase II contains a
stretch of seven amino acids that are repeated
tandemly (52 times in the mouse enzyme and 26
times in yeast)
This heptapeptide has the sequence Tyr-Ser-Pro-
Thr-Ser-Pro-Ser and is known as carboxyl
terminal domain or CTD
These repeats are essential for viability
The CTD sequence may be phosphorylated at the
serines and some tyrosines
In vitro studies have shown that the CTD is
unphosphorylated at transcription initiation, but
phosphorylation occurs during transcription
elongation as the RNA polymerase leaves the
promoter
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TRANSCRIPTION FACTORS
RNA polymerase II is the central enzyme in the
synthesis of RNA from DNA
The enzyme is made up of 12 subunits ( 10-220kD),
in addition it requires an array of other proteins
called transcription factors in order to form active
transcription complex all of which are present in
the nucleus.
These are actually required for basal
transcription initiation
They are highly conserved in all eukaryotes
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TRANSCRIPTION FACTORS
TFIID: It is a complex made up of the TATA-binding
protein (TBP) and at least 12 TBP-associated
factors or TAFs.
TBP is a monomeric protein having a highly
conserved C-terminal domains of 180 residues which
makes contact with the minor groove in the region of
TATA box.
Binding of TBP deforms the DNA so that it bends into
the inside of the saddle & unwound resulting in about
45o kink between the first two and last two base
pairs of 8bp TATA box.
The TAFs are intriguing proteins that appear to play a
variety of role during initiation of transcription
and also during other events that involve assembly of
multiprotein complexes onto the genome
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TRANSCRIPTION FACTORS
TFIIA: It is made up of three subunits of 9, 12 & 35
kD
It binds to TFIID and enhances its binding to the
TATA box by stabilizing the TFII-DNA complex and
also by counteracting the effects of inhibitory
factors
TFIIB: It is single polypeptide of 35kD molecular
weight
It act as a bridging factor allowing the recruitment
and correct positioning of the RNA polymerase to
the transcription start site
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TRANSCRIPTION FACTORS
TFIIF: It is heterodimer of 30 & 74kD subunits
It functions in association with TFIIB to target RNA
polymerase to its promoters by reducing the binding
of polymerase to non-specific sites
TFIIE: It is also a heterodimer of 34 & 57 kD
subunits
It acts as intermediate in recruitment of TFIIH and
also modulates its function
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TRANSCRIPTION FACTORS
TFIIH: It is a complex of about 12 subunits (35-89
kD)
It has three important activities associated with it
Helicase activity is responsible for the opening of the
promoter for the docking of RNA polymerase II to it
A kinase activity in one of its subunits phosphorylates
Carboxyl Terminal Domain of the RNA polymerase II
which is necessary for the promoter clearance as it
brings about the necessary conformational change in its
structure and thus initiating transcription
Thirdly it also functions as a repair enzyme by recruiting
nucleotide exicision repair complex to the site of DNA
lesion
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INITIATION
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QUESTIONS?