MOLECULAR BIOLOGY

TRANSCRIPTION

PROCESS

Dr. Aga Syed SameerCSIR Lecturer (Demonstrator)

Department of Biochemistry,

Medical College,

Sher-I-Kashmir Institute of Medical Sciences,

Bemina, Srinagar, Kashmir, 190018. India.

PROMOTERS

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)

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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INITIATION

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TRANSCRIPTION TERMINATION

Factor-Independent or Intrinsic termination:

It is mediated by the intrinsic sequences of the

transcripts that are being synthesized which cause

them to adopt particular secondary structures which

pauses the RNA polymerase at the end and dissociated

DNA-RNA hybrid

There are two common terminator motifs that have

been found to play role in intrinsic termination, one is

the GC rich inverted repeat centered 15-20

nucleotides before the end of the RNA strand and

other is the polyU sequence at the 3 -׳ end of the RNA

TRANSCRIPTION TERMINATION

These features suggest the following as elements of the

termination mechanism:

1. RNA Polymerase slows down, or pauses, when it

reaches the first GC-rich segment, because the

stability of G-C base pairs makes the template hard to

unwind

2. The pausing gives time for the complementary GC-

rich parts of the nascent transcript to base-pair

with one another. In the process, the downstream

GC-rich segment of the transcript is displaced

from its template (or from that part of the enzyme

molecule to which it is bound). Hence, the ternary

complex of RNA polymerase, DNA template, and

RNA is weakened

TRANSCRIPTION TERMINATION

Factor-Dependent or Extrinsic termination:

It requires a protein called ρ (rho). The ρ protein, a hexamer composed of identical subunits, has been characterized as an RNA-DNA helicase and contains a nucleoside triphosphatase activity that is activated by binding to polynucleotides

It apparently acts by binding to the nascent RNA transcript at a specific cytidine-rich but guanosine-poor site near the 3' end about 50-100 nucleotides in length. When RNA polymerase has paused, It then moves along the transcript toward the 3' end, with its intrinsic helicase activity unwinding the 3' end of the transcript from the template

TRANSCRIPTION TERMINATION

Rho protein facilitates the termination by wrapping

the nascent RNA chain around itself, thereby

destabilizing the RNA-DNA hybrid and

terminating transcription. Approximately 80

nucleotides of RNA wrap around the rho protein.

Rho can also bind to the NusA and other proteins,

this association causes the polymerase to change the

conformation which decreases its interaction with

the DNA and hence causes the polymerase

dissociation from the template DNA

Also, called as Hot Pursuit Model of Termination

TRANSCRIPTION TERMINATION

TRANSCRIPTION TERMINATION

Termination of mRNA transcription is different

in eukaryotes than in prokaryotes

The eukaryotic RNA polymerase II usually continues

to transcribe well past the end of the gene

After the end of the gene has been reached, RNA

polymerase II passes through one or more AATAAA

sequences, which lie beyond the 3' end of the coding

region

The pre-mRNA, carrying this signal as AAUAAA, is

then cleaved by a special endonuclease that

recognizes the signal and cuts at a site 11 to 30

residues to its 3' side

A tail of polyriboadenylic acid, poly(A), as much

as 200 bases long, is added by a special non-template-

directed polymerase

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TRANSCRIPTION INHIBITORS

Actinomycin D: The planar portion of this molecule

inserts (intercalates) into the double helical DNA

between successive GC base pairs, deforming the DNA

This prevents movement of the polymerase along

the template and hence inhibits RNA elongation in

intact cells as well as in cell extracts, it is used to

identify cell processes that depend on RNA synthesis

Acridine inhibits RNA synthesis in a similar fashion

Rifampicin inhibits bacterial RNA synthesis by

binding to the β’ subunit of bacterial RNA

polymerases, preventing the promoter clearance step

of transcription. It is sometimes used as an antibiotic

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TRANSCRIPTION INHIBITORS

α-Amanitin: The mushroom Amanita phalloides has

evolved a very effective defense mechanism against

predators

It produces α-amanitin, which disrupts mRNA

formation in animal cells by blocking Pol II and, at

higher concentrations, Pol III

Neither Pol I nor bacterial RNA polymerase is

sensitive to α-amanitin nor is the RNA polymerase II of

A. phalloides itself.

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APOB MODIFICATION

ApoB is primary apolipoprotein of LDL which is

responsible for carrying cholesterol to tissues

Acts as ligand for LDL receptors in various cells

throughout the body

It is present in two forms – APOB48 & APO100

APOB48 is synthesized exclusively in small intestines

APOB100 is synthesized in liver

Both are coded by APOB gene which produces a

single mRNA transcript larger than 16kb in size

However, the two isoforms are produced as a result of

mRNA editing

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APOB MODIFICATION

The special stop codon is created within the coding

sequence of APOB mRNA at codon 2153

The creation of this special stop codon before the

actual stop codon causes the premature

termination of ApoB during translation

Hence, a short form of ApoB protein is created

APOB48

APOB48 & 100 have common N terminal sequence

APOB48 lacks C-terminal LDL receptor binding

region

APB48 constitutes only 48% of APOB100

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APOB MODIFICATION

The enzyme responsible for this special stop codon

editing is – Apolipoprotein B-mRNA Editing-

enzyme Catalytic Polypeptide

The codon 2153 gets changed from CAA (the actual

stop codon) to UAA (special stop codon);

Glutamine codon to Non sense codon

Hence, the APOBEC-1 catalyze the Deamination

Reaction

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QUESTIONS?