OUTLINE OF
PROKARYOTIC
TRANSCRIPTION
MRS POONAM SINGH
ASSISTANT PROFESSOR
PG DEPARTMENT OF ZOOLOGY
TRANSCRIPTION AN OVERVIEW
Transcription is the enzymic synthesis of RNA on a DNA
template.
This is the first stage in the overall process of gene
expression and ultimately leads to synthesis of the protein
encoded by a gene.
Transcription is catalyzed by an RNA polymerase which
requires a dsDNA template as well as the precursor
ribonucleotides ATP, GTP, CTP and UTP
RNA synthesis always occurs in a fixed direction, from the
5’- to the 3’-end of the RNA molecule .
Usually, only one of the two strands of DNA becomes
transcribed into RNA. One strand is known as the
sense strand.
The sequence of the RNA is a direct copy of the
sequence of the deoxynucleotides in the sense strand
(with U in place of T).
The other strand is known as the antisense strand.
This strand may also be called the template strand
since it is used as the template to which
ribonucleotides base-pair for the synthesis of the RNA.
FORMATION OF
PHSOPHODIESTER BOND IN
TRANSCRIPTION
INITIATION
Initiation of transcription involves the binding of an RNA
polymerase to the dsDNA. RNA polymerases are usually
multi subunit enzymes.
They bind to the dsDNA and initiate transcription at sites
called promoters (Fig. 2).
Promoters are sequences of DNA at the start of genes, that is
to the 5’-side (upstream) of the coding region.
Sequence elements of promoters are often conserved
between different genes.
Differences between the promoters of different genes give
rise to differing efficiencies of transcription initiation and are
involved in their regulation
The short conserved sequences within promoters are the
sites at which the polymerase or other DNA-binding proteins
bind to initiate or regulate transcription.
In order to allow the template strand to be used for base
pairing, the DNA helix must be locally unwound. Unwinding
begins at the promoter site to which the RNA polymerase
binds.
The polymerase then initiates the synthesis of the RNA
strand at a specific nucleotide called the start site
(initiation site).
This is defined as position +1 of the gene sequence .
The RNA polymerase and its co-factors, when assembled on
STRUCTURE OF A TYPICAL TRANSCRITION UNIT
SHOWING PROMOTER AND TERMINATOR AND THE
RNA PRODUCT
ELONGATION
The RNA polymerase covalently adds ribonucleotides to the 3-end
of the
growing RNA chain .
The polymerase therefore extends the growing RNA chain in a
5’ 3’ direction. This occurs while the enzyme itself moves in a 3’
5’ direction along the antisense DNA strand (template).
As the enzyme moves, it locally unwinds the DNA, separating
the DNA strands, to expose the template strand for ribonucleotide
base pairing and covalent addition to the 3’-end of the growing
RNA chain.
The helix is reformed behind the polymerase.
The E. coli RNA polymerase performs this reaction at a rate of
around 40 bases per second at 37°C.
TERMINATION
The termination of transcription, namely the dissociation of
the transcription complex and the ending of RNA synthesis,
occurs at a specific DNA sequence known as the
terminator
These sequences often contain self-complementary
regions which can form a stem–loop or hairpin secondary
structure in the RNA product .
These cause the polymerase to pause and subsequently
cease transcription.
Some terminator sequences can terminate transcription
without the requirement for accessory factors, whereas
other terminator sequences require the rho protein as an
accessory factor.
In the termination reaction, the RNA–DNA hybrid is
separated allowing the reformation of the dsDNA, and the
RNA polymerase and synthesized RNA are released from
the DNA.
RNA HAIRPIN STRUCTURE
E.COLI RNA POLYMERASE
The E. coli RNA polymerase is one of the largest enzymes in the cell.
The enzyme consists of at least five subunits.
ᵝ σ
These are the alpha (α ), beta ( ), beta prime (ᵝ’), omega (ω) and sigma ( )
subunits. In the complete polymerase called the holoenzyme, there are two
subunits and one each of the other four subunits .
The complete enzyme is required for transcription initiation.
However, the σ factor is not required for transcription elongation and is released
from the transcription complex after transcription initiation.
The remaining enzyme, which translocate along the DNA, is known as the core
enzyme and has the structure α 2 ᵝ ᵝ’ ω.
The E. coli RNA polymerase can synthesize RNA at a rate of around 40 nt per sec
at 37°C and requires Mg2+ for its activity.
The enzyme has a non spherical structure with a projection flanking a cylindrical
channel.
The size of the channel suggests that it can bind directly to 16 bp
of DNA. The whole polymerase binds over a region of DNA covering
around 60 bp.
Although most RNA polymerases like the E. coli polymerase have a
multi subunit structure, it is important to note that this is not an
absolute requirement.
The RNA polymerases encoded by bacteriophages T3 and T7
are single polypeptide chains which are much smaller than the
bacterial multi subunit enzymes.
They synthesize RNA rapidly (200 nt per sec at 37°C) and recognize
their own specific DNA-binding sequences.
ALPHA α Subunit
Two identical α subunits are present in the core RNA
polymerase enzyme.
The subunit is encoded by the rpoA gene. The α subunit is
required for core protein assembly, but has had no clear
transcriptional role assigned to it.
When phage T4 infects E. coli the subunit is modified by
adenosine diphosphate (ADP) ribosylation of an arginine.
This is associated with a reduced affinity for binding to
promoters, suggesting that the subunit may play a role in
promoter recognition.
ᵝ
Subunit
One ᵝ subunit is present in the core enzyme.
This subunit is thought to be the catalytic center of
the RNA polymerase.
Strong evidence for this has come from studies with
antibiotics which inhibit transcription by RNA
polymerase.
The important antibiotic rifampicin is a potent
inhibitor of RNA polymerase that blocks initiation
but not elongation.
This class of antibiotic does not inhibit eukaryotic
polymerases and has, therefore, been used
medically for treatment of Gram-positive bacteria
infections and tuberculosis
Rifampicin has been shown to bind to the ᵝ subunit.
Mutations that give rise to resistance to rifampicin map
to rpoB, the gene that encodes the ᵝ subunit.
A further class of antibiotic, the streptolydigins, inhibit
transcription elongation, and mutations that confer
resistance to these antibiotics also map to rpoB gene.
These studies suggest that the ᵝ subunit may contain
two domains responsible for transcription initiation and
elongation.
ᵝ’Subunit
One ᵝ’ subunit is present in the core enzyme. It is encoded
by the rpo C gene.
This subunit binds two Zn2+ ions which are thought to
participate in the catalytic function of the polymerase.
A polyanion, heparin, has been shown to bind to the ᵝ’
subunit. Heparin inhibits transcription in vitro and also
competes with DNA for binding to the polymerase.
This suggests that the ᵝ’ subunit may be responsible for
binding to the template DNA.
The omega( ω) subunit of the RNA polymerase
Omega (omega) is the smallest subunit of bacterial
RNA polymerase (RNAP). Although identified early in
RNAP research, its function remained ambiguous
and shrouded by controversy for a considerable
period.
It has subsequently been shown that the protein
has a structural role in maintenance of the
conformation of the largest subunit, beta', and
recruitment of beta' to the enzyme assembly.
Sigma Factor σ
The most common sigma factor in E. coli is σ
Binding of the σ factor converts the core RNA polymerase
enzyme
into the holoenzyme.
The σ factor has a critical role in promoter recognition, but is not
required for transcription elongation.
The factor contributes to promoter recognition by decreasing the
affinity of the core enzyme for nonspecific DNA sites by a factor
of 10000 and increasing affinity for the promoter
Many prokaryotes (including E. coli) have multiple σ factors.
They are involved in the recognition of specific classes of
promoter sequences.
The σ factor is released from the RNA polymerase when
the RNA chain reaches 8–9 nt in length.
The core enzyme then moves along the DNA synthesizing
the growing RNA strand.
The σ factor can then complex with a further core enzyme
complex and re-initiate transcription.
There is only 30% of the amount of σ factor present in the
cell compared with core enzyme complexes.
Therefore only one-third of the polymerase complexes can
exist as holoenzyme at any one time.
