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?