1.Definition
2.Transcription is selective
3.Transcription in Prokaryotes
•Initiation
•Elongation
•RNA polymerase vs DNA polymerase
•Termination
4.Transcription in Eukaryotes
•Initiation
•Elongation
•Termination
•Post transcriptional modifications
2. Transcription is a process in which
ribonucleic acid (RNA) is synthesized from
DNA.
Transcription is Selective:
•The entire molecule of DNA is not expressed
in transcription.
•RNAs are synthesized only for some selected
regions of DNA.
There exist certain differences in the
transcription between prokaryotes and
eukaryotes.
3. Transcription in Prokaryotes:
Initiation:
The binding of the enzyme RNA polymerase to DNA is the prerequisite
for the transcription to start. The specific region on the DNA where the
enzyme binds is known as promoter region.
A single enzyme—RNA polymerase— synthesizes all the RNAs in
prokaryotes.
RNA polymerase of E. coli is a complex holoenzyme with five
polypeptide subunits— 2α, 1β and 1β’ and one sigma (σ) factor. The
enzyme without sigma factor is referred to as core enzyme.
There are two base sequences on the coding DNA strand which the
sigma factor of RNA polymerase can recognize for initiation of
transcription.
1. Pribnow box (TATA box): 6 nucleotide bases (TATAAT),10 bases
upstream to the starting point of transcription.
2. The ‘-35’ sequence: base sequence TTGACA
RNA polymerase of E. Coli.
4. Transcription in Prokaryotes:
Elongation:
As the holoenzyme, RNA polymerase recognizes the promoter region, the
sigma factor is released and transcription proceeds.
RNA is synthesized from 5′ end to 3′ end (5’→3′) antiparallel to the DNA
template.
The genetic information stored in DNA is expressed through RNA. For this
purpose, one of the two strands of DNA serves as a template (non-coding
strand or sense strand) and produces working copies of RNA molecules.
The other DNA strand which does not participate in transcription is referred
to as coding strand or antisense strand.
RNA polymerase utilizes ribo-nucleotide triphosphates (ATP, GTP, CTP and
UTP) for the formation of RNA. For the addition of each nucleotide to the
growing chain, a pyrophosphate moiety is released. The sequence of
nucleotide bases in the mRNA is complementary to the template DNA strand.
5. RNA polymerase vs DNA polymerase
(RNA polymerase differs from DNA polymerase in two aspects. No
primer is required for RNA polymerase and, further, this enzyme
does not possess endo- or exonuclease activity. Due to lack of the
latter function (proof-reading activity), RNA polymerase has no
ability to repair the mistakes in the RNA synthesized.
This is in contrast to DNA replication which is carried out with high
fidelity. It is, however, fortunate that mistakes in RNA synthesis are
less dangerous, since they are not transmitted to the daughter cells.
The double helical structure of DNA unwinds as the transcription
goes on, resulting in supercoils. The problem of supercoils is
overcome by topoisomerases. )
6.
7. Termination:
The process of transcription stops by termination signals. Two types of
termination are identified.
1. Rho (ρ) dependent termination:
•A specific protein, named ρ factor, binds to the growing RNA (and not
to RNA polymerase) and in the bound state it acts as ATPase and terminates
transcription and releases RNA.
•The ρ factor is also responsible for the dissociation of RNA polymerase
from DNA.
2. Rho(ρ) independent termination:
•The termination in this case is brought about by the formation of
hairpins of newly synthesized RNA. This occurs due to the presence of
palindromes.
Transcription in Prokaryotes:
9. RNA synthesis in eukaryotes is a much more complicated process than the transcription in prokaryotes.
RNA Polymerases:
1. RNA polymerase I is responsible for the synthesis of precursors for the large ribosomal RNAs.
2. RNA polymerase II synthesizes the precursors for mRNAs and small nuclear RNAs.
3. RNA polymerase III participates in the formation of tRNAs and small ribosomal RNAs.
Besides the three RNA polymerases found in the nucleus, there also exists a mitochondrial RNA
polymerase in eukaryotes. The latter resembles prokaryotic RNA polymerase in structure and function.
Promoter Sites:
1.Hogness box (or TATA box)- 25 nucleotides away upstream from the starting site of mRNA synthesis.
2. CAAT box-between 70 and 80 nucleotides upstream from the start of transcription.
Etc.
Transcription in Eukaryotes:
10. Initiation:
RNA Pol II does not contain a subunit similar to the prokaryotic factor, which can
recognize the promoter and unwind the DNA double helix. In eukaryotes, these
two functions are carried out by a set of proteins called general transcription
factors.
The RNA Pol II is associated with six general transcription factors, designated as
TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH.
TFIID consists of TBP (TATA-box binding protein). The role of TBP is to bind the
core promoter.
The transcription factor which catalyzes DNA melting is TFIIH (Helicase) .
However, before TFIIH can unwind DNA, the RNA Pol II and at least five general
transcription factors have to form a pre-initiation complex (PIC).
Transcription in Eukaryotes:
11. Elongation: (same as prokaryotes)
The carboxyl-terminal domain (CTD) of the largest subunit of RNA Pol II is critical
for elongation. During elongation it has to be phosphorylated by TFIIH with kinase
activity.
Transcription in Eukaryotes:
12. Termination:
In the case of protein-encoding genes, the cleavage site which determines the “end” of
the synthesizing mRNA occurs between an upstream AAUAAA sequence and a
downstream GU-rich sequence separated by about 40-60 nucleotides in the
synthesizing mRNA.
Once both of these sequences have been transcribed, a protein called
CPSF(Cleavage Polyadenylation Specificity Factor) in humans binds the AAUAAA
sequence and a protein called CstF(Cleavage stimulating Factor) in humans binds
the GU-rich sequence. These two proteins form the base of a complicated protein
complex that forms in this region before CPSF cleaves the nascent pre-mRNA at a site
10-30 nucleotides downstream from the AAUAAA site.
The Poly(A) Polymerase enzyme which catalyzes the addition of a 3′ poly-A
tail(Polyadenylation) on the pre-mRNA.
Transcription in Eukaryotes:
14. Post-transcriptional Modifications
This process is required to convert the RNAs into the active forms.
A group of enzymes, namely ribonucleases, are responsible
for the processing of tRNAs and rRNAs of both prokaryotes and
eukaryotes.
Messenger RNA:
•The primary transcript of mRNA is the hnRNA in eukaryotes, which is
subjected to many changes before functional mRNA is produced.
1. The 5′ capping:
The 5′ end of mRNA is capped with 7-methylguanosine by an unusual
5’→5′ triphosphate linkage. S-Adenosylmethionine is the donor of
methyl group. This cap is required for translation, besides stabilizing the
structure of mRNA.
15. 2. Poly-A tail:
A large number of eukaryotic mRNAs possess an
adenine nucleotide chain at the 3′-end. This
polyadenylation is a Template independent
polymerization of mRNA. It occurs to stabilize
mRNA. However, poly-A chain gets reduced as the
mRNA enters cytosol.
Post-transcriptional Modifications
16. 3. Introns and their removal:
Introns are the intervening nucleotide sequences in mRNA
which do not code for proteins.
Exons of mRNA possess genetic code and are responsible for
protein synthesis.
The removal of introns is promoted by small nuclear
ribonucleoprotein particles (snRNPs). snRNPs, (pronounced
as snurps) in turn, are formed by the association of small
nuclear RNA (snRNA) with proteins.
The term spliceosome is used to represent the snRNP
association with hnRNA at the exon-intron junction.
Post-transcriptional modifications of mRNA occurs in the
nucleus. The mature RNA then enters the cytosol to perform
its function (translation).
Post-transcriptional Modifications
