Transcription
Intro
Transcription is defined as the synthesis of RNA molecules using DNA as a template, that
results in the transfer of the information stored in double stranded DNA into single stranded
RNA.
5’ 3’
3’ 5’ Template strand
5’ 3’ mRNA [ TRANSCRIPTION]
Primary Transcript → post Transcriptional modification → active RNA molecule
Basic requirement
1. Template: single strand of DNA acts as Template strand.
2. Substrate: ATP, GTP, CTP, UTP
3. Enzyme: DNA dependent RNA polymerase called as RNA polymerase
a) Prokaryotic RNA polymerase
It is a holoenzyme with 5 subunits. 2α, 1β, 1β’ & 1 sigma factor.
Sigma factor is required for binding of the polymerase to specific promotor
regions of DNA template.
b) Eukaryotic RNA polymerase
RNA polymerase I
RNA polymerase II
RNA polymerase III
Stages of Transcription
I Initiation
Initiation of transcription involves binding of RNA polymerase (+ sigma factor) to the
template at the promoter site.
Promoter sequences are responsible for directing RNA polymerase to initiate transcription at
a particular point known as Initiation site.
Prokaryotic promoters
a) Pribnow box (TATA)
Consist of –TATAAT- consensus sequences, with 10 base pairs upstream the
starting point of transcription.
b) -35 sequence:
Consist of –TTGACA- sequences, with 35 base pairs upstream the starting point
of transcription.
Eukaryotic promoters
a) Hogness box
Consist of –TATAAT- sequence.
-25 base pairs upstream to start point.
b) CAAT box
Consist of – GGCAATCT- sequences.
-75 base pairs upstream to start point.
c) GC box
Consist of – GGGCG - sequences.
Found between -70 & -80 upstream to start point.
d) Enhancers increase the rate of transcription.
Silencers decrease the rate of transcription.
Steps in Initiation:
1. RNA polymerase → sigma factor recognizes the promoter region and binds with
promoter site of DNA forming ‘Pre-initiation complex’.
2. Unwinding of the DNA helix.
3. First nucleotide complementary to the base present in DNA, attaches to the initiation
site on beta subunit of RNA polymerase.
4. RNA polymerase catalyses formation of phosphodiester bond between first 2 bases.
5. Enzyme moves to the next base on DNA template.
Elongation
1. RNA polymerase utilises ATP, GTP, CTP, UTP for the formation of RNA.
2. As 10 nucleotides are added, sigma factor dissociates.
3. The core enzyme continues the elongation of the transcript, until a termination signal
is reached.
4. RNA polymerase
- Doesn’t require primer
- Moves synthesizing 5’ to 3’ direction
- Obeys Base pairing rule.
- No nuclease activity & no proof reading activity.
Termination
a) Rho- dependent termination
- Rho factor, a specific protein factor binds to the growing RNA at Rho utilisation
sites (rut sites).
- Recognises termination signal
- Its ATP- dependent Helicase activity displaces RNA polymerase → Termination
of RNA synthesis.
b) Rho-independent termination
- Involves formation of Hairpin loop with invert repeat GC rich sequences in newly
synthesized RNA. This loop blocks the RNA polymerase.
- Hair loop is followed by sequence of uracil residues. The A-U bonds are very
weak resulting in release of the newly synthesized RNA.
- RNA transcription ends.
Post-transcriptional modification
▪ Def: Primary transcript made by RNA Polymerase undergoes further enzymatic
alteration called post-transcriptional processing.
▪ mRNA formed and released from the DNA template in known as ‘Primary transcript’
[Hetero nuclear hn RNA].
▪ Prokaryotic post-transcriptional processing.
1. Cleavage of large precursor of RNA – removal of excess sequences from the
primary transcript by endonucleases or exonucleases.
2. Terminal addition of nucleotides eg. CCA sequence at 3’ end of tRNA.
3. Nucelotide modification eg. Uridylate residues modified to form ribothymidylate
& pseudouridylate.
▪ Eukaryotic post-transcriptional processing
▪ Undergoes extensive processing to become mature RNA.
1. Endonuclease cleavage:
Large precursor RNA → excess sequences are cleaved from it.
Eg. Processing of ribosomal RNA
2. Poly A tail
- 3’ end is polyadenylated.
- Possess (20 – 250) adenine nucleotides.
- Importance: Protects mRNA, stabilizes and determines half-life of mRNA.
3. Capping at 5’ end
- mRNA is capped at 5’ end by 7-methyl guanosine triphosphate.
- S-Adenosyl Methionine is the methyl donor.
- Importance: Stabilises mRNA and recognition of mRNA by translating
machinery.
4. Methylation
- Methylation of N6 of adenine and 2’-hydroxy group of ribose.
5. Splicing
- Exons are the coding sequence of the gene.
- Introns are the non-coding sequence or intervening sequences between exons.
- Def: The process by which introns are excised and exons are linked to form the
functional mRNA is called as Splicing.
- Formation of Spliceosome:
a) Small nuclear RNA eg. U1, U2, U4, U5,U6,U7.
b) It recognises 5’ & 3’ splice site of introns.
c) Formation of looped structure.
d) Cleavage & removal of introns.
e) The exons are joined by 3’ 5’ phosphodiester linkage.
Inhibitors of Transcription
- Actinomycin D &Mitomycin: Intercalate with DNA strand, thus blocking
transcription.
- Rifampicin: (Anti-Tuberculosis) Binds to β subunit of RNAP.
- α-amanitin: (Mushroom poison) Inactivates Eukaryotic RNAP II.
- 3’ deoxy adenosine: incorrect entry of nucleotide results in termination.
