TRANSCRIPTION AND RNA PROCESSING
Transcription And Post-transcriptional
Processing In Prokaryotes
Transcription is defined as the synthesis of RNA molecule
using DNA as a template that results in the transfer of
the information stored in double-stranded DNA into a
single-stranded RNA; which is used by the cell to direct
the synthesis of proteins.
Cellular RNAs include:
Messenger RNA (mRNA)
Ribosomal RNA (rRNA)
Transfer RNA (tRNA)
Several small nuclear RNAs (snRNAs).
All are transcribed from DNA. The first three RNAs are
involved in protein synthesis and snRNA is involved in
mRNA splicing.
Basic Requirements for Replication
Template
Substrates
Enzymes
Template
A single-strand of DNA acts as a
The DNA strand that is transcribed to RNA molecule is
referred to as template strand.
The other DNA strand is referred to as the coding
strand of the gene.
Substrate
1. ATP
2. GTP
3. CTP
4. UTP
Enzyme
DNA dependent RNA polymerase, called RNA
polymerase (RNAP).
Prokaryotes have single RNA polymerase (RNAP) that
transcribes all three RNAs, i.e. mRNA, rRNA, and
tRNA
Eukaryotes have three RNA polymerases, designated I, II
and III.
1. RNA Polymerase I: Synthesis of rRNA.
2. RNA Polymerase II: Synthesis of m-RNA and sn RNA.
3. RNA Polymerase III: Synthesis of tRNA.
4. Mitochondrial RNA polymerase (mtRNAP): Synthesis
of all the three types of RNA in mitochondria.
RNA polymerase (RNAP)
RNAP contains five subunits (2α, β′, β, ω) which form
the core enzyme.
The active enzyme, the holoenzyme contains core
enzyme and a sixth subunit called sigma (σ) factor.
σ subunit binds to core enzyme and directs enzyme to
specific binding sites on the DNA.
RNA polymerases lack proofreading 3′ to 5′
exonuclease activity as that of DNA polymerases.
RNA polymerase requires Mg2+ as well as Zn2+ for
its activity.
Components of prokaryotic holoenzyme RNA
polymerase.
Stages of Transcription
1. Initiation
2. Elongation
3. Termination
Initiation
Initiation of transcription involves binding of RNA
polymerase (core enzyme + σ factor) to DNA
template at promoter site.
The sigma factor facilitates the RNA polymerase to
recognize and bind to promoter sequences.
Promoters are characteristic sequences of DNA which
are different in prokaryotes and eukaryotes
Prokaryotic genes have two promoter sequences.
1.Pribnow box (–10 region) has the nucleotide sequence
TATAAT, found 10 base pairs away from
(upstream) the start point.
2. –35 region, has nucleotide sequence TTGACA, found
35 base pairs away from (upstream) the start point.
Prokaryotic promoter sites for
transcription.
Promoter sequences directs RNA polymerase to
initiate transcription at a particular point known
as start point or initiation site.
The binding of the RNA polymerase to the DNA
template results in unwinding of DNA double
helix.
RNA polymerase then begins to synthesize a
transcript of DNA sequence and short piece of RNA
is made.
As with replication, transcription is always in the 5′
to 3′ direction.
The first base is usually a purine nucleotide.
By the time 10 nucleotides have been added, the σ
factor dissociates
Elongation
Sigma is released and the RNAP move along
the template stand and continues the elongation
of the transcript.
The process of elongation of the RNA chain
continues until a termination signal is reached
Figures A to E: Process of transcription in prokaryotes
A: Recognition of promoter by sigma factor
B: Binding of core enzyme and starts the synthesis of RNA
C: Elongation continues until termination region is reached
D: Termination of transcription by Rho factor
E: Newly synthesized RNA (primary transcript)
Recognition of promoter by sigma factor
Binding of core enzyme starts synthesis of RNA
Elongation continues until termination
region is reached
Termination
In prokaryotes, termination of transcription
occurs by one of the two mechanisms:
1. Rho-dependent
2. Rho-independent.
Termination of transcription by Rho factor
Newly synthesized RNA
A hairpin loop structure followed by a sequence of
uridine residues at the 3’ end of an mRNA transcript
Post-transcriptional Processing
The RNAs formed during transcription are called
primary transcript. The primary transcript
normally undergo further enzymatic alteration,
called post- transcriptional processing.
Post-transcriptional processing is required to
convert the primary RNAs into functional or
active forms.
Processing may involve either:
Cleavage of large precursor of RNA to a
smaller molecule
Splicing
Terminal addition of nucleotide
Nucleoside modifications.
m-RNA processing
In prokaryotes mRNA is not post-transcriptionally
processed. Prokaryotic mRNA is functional
imme- diately upon synthesis.
In eukaryotes the primary transcript of mRNA is
hnRNA (heterogeneous nuclear RNA).
After transcription hnRNA is extensively
modified to form functional mRNA.
1. 5'-capping:
The 5' end of eukaryotic mRNA is capped with
7-methylguanylate.
2. Addition of poly A tail:
The 3' end of most eukaryotic mRNAs posses a
chain of 200–300 adenine nucleotides and called
Poly A tail. Poly A tail is not transcribed by DNA
but rather is added after transcription.
3. Removal of introns
Introns are nucleotide sequences on mRNA
that do not code for proteins. Exons are the
coding sequences that code for proteins.
The process by which introns are excised and
exons are linked to form functional mRNA is
called splicing.
Inhibitors of Transcription
Rifampin: It is an anti-tuberculosis drug, which
inhibits the initiation of transcription by binding
b-subunit of prokaryotic RNA polymerase.
Rifampin has no effect on eukaryotic nuclear
RNA poly- merases.
Dactinomycin (Actinomycin D): Dactinomycin is
a therapeutic agent in the treatment of some
cancer. It binds tightly and specifically to double
helical DNA and thereby prevents the movement
of the RNA polymerase.
REVERSE TRANSCRIPTION
Certain RNA viruses that infect animal cells carry
within the viral particle an RNA-dependent DNA
polymerase called reverse transcriptase.
The RNA viruses that contain reverse transcriptases are
known as retroviruses (retro is the Latin prefix for
“backward”) because the genetic information flows
from RNA to DNA rather than from DNA to RNA.
Reverse transcriptase produces DNA from viral RNA.
Extension of the central dogma to include RNA dependent synthesis
of RNA and DNA.
Some retroviruses, classified as RNA tumor viruses,
contain an oncogene that can cause the cell to grow
abnormally.
The first retrovirus of this type to be studied was the
Rous sarcoma virus.
Another important class of retroviruses includes
human immunodeficiency virus (HIV), identified in
1983, which causes acquired immunodeficiency
syndrome (AIDS).
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