Transcription is a cellular process during which RNA is

synthesized
using DNA as a template. All three types of cellular RNAs
are copied from DNA template in the process of transcription.
Only mRNA contains instructions for protein synthesis.
The other two types (rRNA and tRNA) are not translated,
but perform other vital functions during protein synthesis.
Transcription is similar to replication in terms of
chemical mechanism, polarity (direction of synthesis)
and use of a template. It differs from replication in two
important aspects:
1. Transcription does not require a primer.
2. It involves only short segments of DNA. Within these
segments, one of the separated strands of DNA serves
as a template (i.e. the template strand); the other
strand of DNA is the coding strand.
RNA Polymerase (RNAP)
RNA polymerase (RNAP) is the principal enzyme of
transcription
in prokaryotes, which synthesizes all three
types of cellular RNAs—mRNA, tRNA and rRNA—in
E. coli. It acts according to instructions given by a DNA
template
and does not need a primer. It is a complex enzyme
(500 kD), consisting of various subunits. The subunit
structure of the holoenzyme is _2 ____ (Table 22.2).
The sigma (_) subunit is loosely bound to the other subunits.
It is not required for the catalytic activity, but it
enables the RNA polymerase to initiate the transcription
by recognizing certain specifi c sequences on the DNA.
Thereafter it gets dissociated from the holoenzyme, leaving
behind the core enzyme (_2 ___).
Each of the subunits of the holoenzyme has a distinct
role. The __-subunit helps in the binding of DNA template;
while the _-subunit binds the ribonucleoside triphosphate
substrates. The _-subunit is necessary to reconstitute active
enzyme from separated subunits. The _-subunit plays
an important role in initiating transcription.
A. Three Stages of Transcription
Transcription occurs in three stages, i.e. initiation, elongation
and termination.
Initiation
Before starting transcription, RNAP has to fi nd the gene.
Genes possess recognition sequence immediately upstream
(i.e. on 5_ side) of the sequence that would be transcribed
(Fig. 22.1). Transcription is initiated when RNAP interacts
with the recognition sequences on the coding strand of
DNA, called the promoters. Binding of the RNAP to promoter
directs transcription of the adjacent segment of DNA.
How does the enzyme fi nd the promoter? The _2____
holoenzyme fi rst positions itself on the duplex DNA,
forming transient hydrogen bonds with exposed base
pairs. This binding is non-selective, occurring with a
moderate affi nity between the holoenzyme and the
bases. Then the holoenzyme slides along DNA. As it
encounters the promoter sequence (which is recognized
by the _-subunit), it stops moving further. The transcription
initiation complex is now formed, called the closed
complex; it consists of the _2 ___ _ holoenzyme plus the
duplex DNA. The duplex DNA must be unwound at this
stage, so that one of its strands may serve as template.
This unwinding is brought about by the _-subunit, starting
at the conserved AT-rich sequence (Fig. 22.1). This
sequence, called the Pribnow box (after David Pribnow
who described it in 1975), lies in a region about 10 base
pairs upstream of the site where mRNA synthesis starts
(i.e. transcription initiation site). A segment of nearly 17
base pairs of DNA is unwound in this manner. This
results in formation of the open complex, which consists
of the unwound DNA plus the _2 ___ _ holoenzyme.
Thus, the _-subunit plays major role in initiation of the
transcription by:
1. Permitting RNAP to recognize specifi c initiation site
on the coding strand of DNA.
2. Causing unwinding of the duplex DNA.
Role of _ Subunit
There has been considerable progress
in our understanding of the mechanism by which the
_-subunit helps specifi c recognition. It does so by
decreasing affi nity of the RNAP for the general regions of
DNA by a factor of about 104. Consequently, the relative
affi nity of the RNAP for the promoter region becomes
much higher, and so the two bind readily.
Role of promoter sequences: The promoter sequences are
clustered approximately 10 base pairs (called the –10 bp
sequence) and 35 base pairs (called the –35 bp sequence)
upstream of the transcriptional initiation site. It is noteworthy
that all promoter sequences are recognized by the
same _-subunit, even though the promoters of different
genes do not have identical nucleotide sequence.
However, a consensus sequence of the most commonly
encountered bases can be deduced.
_ The –10 sequence has the consensus TATAAT. It is
named Pribnow box after the discoverer. It is an
important recognition site that interacts with the _
factor of RNA polymerase.
_ The –35 sequence has the consensus TTGAGA and is
important in DNA-unwinding during transcriptional
initiation (Fig. 22.1).
It was puzzling as to why different genes have different
promoters. The most plausible explanation is that different
genes have to be transcribed at different rates. Some
are transcribed up to 10 times per minute, whereas others
are transcribed only once in 10 minutes. The factors
accounting for different rates of transcription are:
(a) affi nity of binding of the RNAP holoenzyme to the
promoter site, and
(b) the rate of transition from the closed to the open
complex. These processes depend on base sequence of the
promoter.
Greater the resemblance between the promoter
and the consensus sequences, faster are the above processes,
and greater in the rate of transcription. The
sequences of strong promoters correspond well with the
sequences shown in Figure 22.1, whereas weaker promoters
have sequences that differ from these.
Despite its key role in initiation, the _-subunit does
not participate in transcription further. It dissociates
from the core enzyme after formation of approximately
10 phosphodiester bonds of the new RNA. The dissociation
of the _-subunit marks the end of the initiation
phase of transcription and sets stage for the next phase of
transcription, i.e. the elongation phase. The dissociated
_-subunit then joins another core polymerase to initiate
another round of transcription. Thus we see that the
holoenzyme is involved in selection and initiation also,
whereas role of the core enzyme is elongation only.
Elongation
After dissociation of the _-subunit, affi nity of the core
enzyme for the promoter decreases markedly. It now
moves along the adjacent base sequences of the DNA. The
core enzyme plays a pivotal role in the elongation phase,
catalyzing the polymerization. New nucleotide units are
incorporated in the nascent RNA, one at a time, according
to the base pairing rule. Thus, A in DNA is transcribed to
U in mRNA, G is transcribed to C, T to A, and C to G. The
polymerization occurs in the 5__3_ direction like in case
of replication. RNAP is processive, i.e. a single enzyme
molecule can remain attached to the template and carry
out transcription till the end. The region containing RNA
polymerase, DNA and nascent RNA is called a transcription
bubble, because it contains locally unwound
(melted) bubble of DNA. The extent of unwinding area
is about 17 base pairs per polymerase molecule (Fig. 22.2).
Nascent RNA forms a hybrid helix with the template
DNA strand in this region.
The precursor molecules for new RNA synthesis are
ribonucleoside triphosphates. The 3_-hydroxyl of the
RNA is so positioned that it attacks the innermost phosphate
of the incoming precursor, forming a new phosphodiester
bond. The pyrophosphate that is released
drives forward the reaction. This is analogous to the
mechanism of “new nucleotide incorporation”, described
in replication (Fig. 21.12).
Length of the DNA-RNA hybrid, and the extent of
unwound area in duplex remains constant as the RNA
polymerase moves along the DNA template. This indicates
that DNA is rewound upstream at the same rate as it is
unwound downstream. Furthermore, as RNA polymerase
causes local unwinding of the duplex DNA, its movement
is associated with generation of waves of positive supercoiling
ahead of it and of negative supercoiling behind it.
Such transcription driven supercoilings are relieved by
action of topoisomerases.
RNA polymerase resembles DNA polymerase in several
ways, but it differs from the latter in two important aspects:
1. It does not require a primer; it can initiate chain synthesis
by assembling the fi rst nucleotide itself.
2. It lacks exonuclease activities; thus, in contrast to DNA
polymerase, it does not edit the nascent polynucleotide
chain. Consequently, the fi delity of transcription
is much lower than that of replication. The error rate
in transcription is 104 to 105 times more than that in
DNA replication. But relatively higher error-rate in
transcription is tolerated since RNA is synthesized
and degraded continuously; thus, a single error causes
little damage. It is of less consequence to the cell
since these errors are not transmitted to the daughter
cell, or to the next generation. By contrast, any error
in replication produces alteration in permanently
stored genetic information, which is transmitted to
progeny.
Comparative features of RNAP and DNAP are given in
Table 22.3.
Sense (_) and antisense (–) strands: It merits re-emphasis
that only one strand of the duplex DNA is copied, directing
synthesis of new RNA in a given region of genome
(Fig. 22.3); it is the template or non-coding strand, also
called the antisense (–) strand. The other strand is sensestand,
also called coding (or non-template) strand. The
RNA produced has same sequence as the the sense (_)
strand (except that T replaces U).
It is evident from what has been described so far, that
nucleic acid synthesis (whether of DNA or RNA) requires
a DNA template. Thus, genetic information coded in
DNA is the prime factor for directing the type of protein
synthesized.
Termination
RNAP must know the defi ned site at which to stop RNA
synthesis, so that the appropriate size of transcript is produced.
This part known as transcription termination is
probably the least understood part of RNA synthesis. It
occurs by two well characterized mechanisms. The fi rst
mechanism, rho-dependent termination, requires the
action of a protein factor, called rho ( ), which recognizes
certain termination signals. This halts movement
of RNAP along the DNA template. The other mechanism
does not require participation of the rho ( ) factor i.e.
-independent termination).