Molecular biology transcription mb 08
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Transcript of Molecular biology transcription mb 08
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?