2. LEARNING OBJECTIVES
After completion of this lecture learner should
be able to define/ Describe:
Synthesis of RNA from DNA template
The primary transcript
DNA dependent RNA polymerase
Steps of RNA synthesis
Post Translational Modification
Synthesis of RNA from DNA template.
The sequence of ribonucleotides in RNA molecule is
complementary to the DNTPs in one strand of DNA
called the template strand or sense strand.
The other strand is called the coding strand or anti
sense strand because it is identical to the RNA except
U for T.
5. Direction & enzyme involved
The information in the template
strand is read in 3’ 5’ direction &
the RNA is synthesized in 5’ 3’
Enzyme involved DNA dependent
RNA polymerase. The enzyme
attaches itself at a specific on the
DNA, the promoter site on the
template strand. This is followed by
initiation of RNA synthesis.
6. The primary transcript
hnRNA generated by RNAP is
promptly capped by 7-methyl
guanosine tri phosphate, which will
eventually appear in the mRNA.
hnRNA contains both introns and
The cap is necessary for protection of
mRNA from the action of 5’
exonuclease, also for recognition of
mRNA by the ribosome
7. DNA dependent RNA
in E coli it exists as a core molecule
having 5 subunits. 2 α, ββ‘ w. The
core RNAP utilizes a protein factor
called sigma factor.
Core enzyme + sigma factor = Holo
9. Steps o RNA synthesis
Initiation binding of holoenzyme to
the template at the promoter site to
form the initiation complex. This is
the closed complex. By sigma factor
there is unwinding of 2 DNA
strands. open complex.
Binding is followed by a
conformational change of the RNAP.
10. Initiation Steps continued
First nucleotide usually a purine
associates with the β subunit of the
enzyme. This becomes the 5’ of the
In the presence of four RNTPs the
RNAP moves to the 2nd base in the
Sigma factor is released.
The enzyme polymerizes the
ribonucleotides in a specific sequence
that is dictated by template strand
and interpreted by Watson and
Crick’s base pairing rules. PPi is
releases at every step.
A purine ribonucleotides is usually
first polymerized into the RNA
Unlike DNAP, RNAP does not require a
primer and does not have proof
RNAP has unwindase activity which
causes local unwinding of the DNA
As the RNAP pushes its way between
the strands it creates a +ve super
coiling ahead and - ve super coiling
behind it. Which are released by DNA
gyrases and topoisomerases
Two types ‘ rho’ dependent and ‘
‘rho’ dependent here termination is
signaled by a sequence in the
template strand of the DNA molecule
(CA rich region) called ‘rut’ (rho
utilization) a signal that is recognized
by rho factor.
Rho protein has an ATP dependent
16. Rho independent
The RNA transcript must be able to
form a stable hairpin turn that slows
down the progress of RNAP. The
hairpin turn is complementary to a
Following the hairpin turn the RNA
transcript must have a string of ‘U’ s.
the A=U bonding is weak which
facilitates its separation from the DNA
After termination the core enzyme
separates from the DNA template.
With the assistance of another sigma
factor the core enzyme recognizes
another promoter at which the
synthesis of a new RNA molecule
19. Transcription of eukaryotic
Far more complicated process than
that in prokaryotic gene.
In addition to RNAP recognizing the
promoter region and initiating RNA
synthesis , a number of transcription
factors bind to distinct sites of DNA.
There are 3 classes of RNAP in the
20. RNAP – classes
RNAP1 synthesizes the 45S
precursor of r RNA (5.8s, 18s, 28s ).
RNAPII synthesizes the precursor of
mRNA and some snRNAs.
RNAPIII synthesizes smaller RNAs
– 5srRNA, tRNA, snRNA.
21. Post transcriptional
A primary transcript is a linear copy
of a transcriptional unit, the segment
of DNA between initiator and
The primary transcript of both tRNAs
and rRNAs are post transcriptionally
modified by cleavage of the original
transcripts by ribonucleases. tRNAs
are further modified.
22. r RNAs and t RNAs
In eukaryotes single 45 s precursor
gives rise to 5.8s, 18s and 28s r
RNAs. 5s rRNA is produced from a
separate precursor molecule.
30 s precursor in prokaryotes
produces 5s 16 s and 23 s r RNAs.
Both pro and eukaryotic t RNAs are
made from longer precursors that
must be trimmed.
23. Messenger RNA
Prokaryotic mRNA is generally
identical to its primary transcript,
whereas eukaryotic mRNA is
extensively modified after
tRNAs and rRNAs of both pro and
eukaryotes are modified after
The cap is 7 methyl guanosine tri
phosphate linked to the 5’ terminal of
the mRNA. The addition is catalyzed
by the enzyme guanylyl transferase.
The methylation of the terminal
guanine is catalyzed by the enzyme 7
methyl transferase S.adenosyl
methionine is the methyl donor.
27. Function of the CAP
Facilitates the initiation of translation.
Helps the recognition of mRNA by the
protein synthesizing machinery
Protects the mRNA from the action of
28. Addition of poly A tail
A chain of 40 – 200 adenine
nucleotides is attached to the 3’ end
of the primary transcript. This is
added by the enzyme poly A
Most eukaryotic mRNAs have poly A
tail except histone mRNA.
Function is to stabilize the mRNA.
After the mRNA enters the cytosol the
poly A tail is gradually shortened.
30. Intron removal
From the primary transcript introns
are removed and the exons are
spliced together to form mature
snRNAs are associated with proteins
to form snRNPs (small nuclear
ribonucleo proteins ) which facilitates
removal of introns and splicing of
After intron removal mature mRNA
leave the nucleus and enter the
32. RNA editing
Central dogma is DNA RNA
Protein. So change in DNA will be
reflected into RNA and into protein,
but sometimes the coding information
can be changed at the RNA level. This
is called RNA editing.
example apo B 100 and apo B 48,
both are synthesized from the same
mRNA by RNA editing.
SNRNP, associated with hnRNA at the
exon- intron junction form
spliceosome. It consists of hnRNA, 5
snRNAs ( U1, U2, U4, U5, U6) and
more than 50 proteins. collectively
they are also called ‘SNURP’.
SNURPs position the RNA segments
for splicing, cut the exon- intron
junction, joins the exons to form a
continuous sequence after intron
They are RNA molecules with catalytic
activity. Several enzymatic actions have
been attributed to RNA.
SnRNA intron removal and splicing.
Peptidyl transferase protein
RNAse P modification of tRNA precursor.
They have specificity and obey M.M.
35. Inhibition of RNA
Actinomycin -D Elongation of RNA
chain is inhibited both in pro and
Rifampicin inhibits bacterial RNAP.
Acridine acts in the same way as
actinomycin – D.
3’ deoxy adenosine causes
premature chain termination.
α- aminitine inhibits RNAPIII.
36. Promoter Region
The promoter sequences are
clustered approximately 10 base pairs
and 35 base pairs upstream (-10 and
– 35 sequences) from the
transcription start site.
5‘TATAAT3’ for the -10 bp sequence
( Prinbow box) and 5’TTGACA’ for –
All promoter sequences are
recognized by the some sigma
subunit of the RNAP.
38. Promoter (eukaryote)
-25 to -30 bp upstream from the
transcription start site 5’TATAAAAG 3’
(TATA or hogness box).
Sequence further upstream G.C box
and CAAT box.
Enhancer and repressor sequence,
both upstream and downstream.