to alter transcription profiles multiple times during cell development. However, transcription does not need
to be extremely accurate, because a cell can still operate fully even if 0.1% of its RNAs are not functional.
In contrast, DNA replication requires that all of the genetic information in the cell be copied, and copied
only once, into a single daughter molecule that is as identical to the parent molecule as possible. DNA
replication is therefore extremely accurate, with rounds of proofreading and error correction, as well as
checks to make sure that a DNA strand only becomes copied once per cell cycle.
6.6.2 Transcription in Prokaryotes
Transcriptioninvolves the copying of a gene into an RNA molecule. Several phases are involved in this
process, namely, initiation of transcription, elongation, terminationand RNA processing(Section 7.2).
There are many similarities between prokaryotes and the eukaryotes in these processes.
6.6.2. 1Prokaryotic RNA Polymerases. There are two types of RNA polymerase. Viral polymerases
are simple, single subunit enzymes in the range of 1 2 104 Da in mass. These polymerases can only
initiate transcription from one or a very small number of very similar promoters. In contrast, all RNA poly-
merases that transcribe cellular genes are large, multi-subunit enzymes, which are more versatile in their
ability to recognise different promoters.
In prokaryotes, a single RNA polymerase is responsible for the synthesis of all RNA. The complete
enzyme (holoenzyme) has a molecular mass approximately 4.8 105 Da. It is pentameric in structure and
is comprised of two a subunits and two related B subunits, b and b, together with an associated unit called
(sigma). There are several sigma factorsavailable^33 and these modulate the specificity of the RNA poly-
merase for different promoters (see below).
6.6.2.2 Prokaryotic Transcriptional Initiation. Transcriptional initiationis the first event in copying
the DNA template into RNA.^34 This occurs at a specific region of the gene, called the promoter. E. coliRNA
polymerase that lacks a sigma factor (the core enzyme) has a relatively weak affinity for all DNA, with no
great preference for promoter regions. The function of is to make sure that RNA polymerase binds stably
to DNA only at promoters. RNA polymerase containing a sigma factor (holoenzyme) has a far lower affin-
ity for DNA in general but a higher affinity for promoter regions in particular. Different promoters show
large differences in their affinities for the holoenzyme, with ‘strong’ promoters having far higher affinities.
6.6.2.2.1 Steps in Prokaryotic Transcriptional Initiation. The process of transcriptional initi-
ation has been elucidated in vitrousing purified RNA polymerase holoenzyme and a DNA template. Four
distinct stages are observed (Figure 6.17). First, the core enzyme binds to a region from about 40 bases
upstream (the35 box) to about20 bases downstream of the transcription start site to form a closed
promoter complex. At this point the DNA template is still an intact double helix. Second, the RNA poly-
merase moves downstream and a limited region of the helix at another conserved sequence (the Pribnow
or10 box) is unwound to form an open promoter complex. Third, the polymerase begins to synthesise a
short RNA molecule on the template DNA strand at the start site. Usually, several abortive short RNAs of
between two and nine nucleotides are synthesised before the polymerase succeeds in clearing the pro-
moter. At this point the factor detaches from the holoenzyme.
6.6.2.2.2 Promoter Identification. Promoters, both prokaryotic and eukaryotic, have been identi-
fied in one or a combination of the following ways:
(i) Consensus searches. Many promoters are aligned with each other and conserved regions are thus
identified. The Pribnow and35 boxes were originally identified in this way.
(ii) Mutation analysis. Naturally occurring or mutagen-induced mutations that affect transcriptional
initiation are examined by sequence analysis of the promoters to determine the molecular basis
for the mutations.224 Chapter 6