(TTGACA) is usually located between 16 and 18 bp
further upstream and is designated the “–35 region.”
Together, the –35 and –10 sequences provide the
necessary information to allow RNAP to identify a
promoter. Specific interactions between the
s
-subunit
of holoenzyme and the –35 and –10 elements allow
RNAP to tightly engage the promoter region. Once
bound, RNAP interacts with an extended region of
DNA, ultimately contacting the DNA over a region of
60 or more base pairs (from 2 40 to þ 20).
Once bound at a promoter site RNAP melts the DNA
over approximately one turn of the helix (from about
2 10 to þ2) to expose the template strand of the DNA.
The process of converting the double-stranded promoter
region DNA into a locally melted structure (called the
open complex) requires the action of
s
-factor to initiate
the melting process and is then driven by the association
of the template strand of the DNA with the RNAP active
site (comprised largely of the
b
-and
b
0
-subunits). The
open complex is then poised to initiate synthesis of an
RNA chain. The region of DNA strand-separation is
often referred to as the transcription bubble, to
distinguish it from the otherwise double-stranded
DNA in the surrounding regions of the genome.
The process of transcription elongation involves the
assembly of ribonucleoside triphosphate precursors into
an RNA chain containing the sequence complementary
to the template DNA. During transcription elongation
the DNA template is threaded through the active
site of RNAP and, as a result, the transcription bubble
is propagated down the DNA with melting of the
DNA occurring as the DNA enters the active site and
reannealing of the template occurring as the DNA exits
the RNAP. The complex containing the DNA template,
RNAP, and the growing RNA chain is often referred to
as the ternary complex. As the growing RNA chain
emerges from RNAP, it is thought to displace the
s
-subunit, and the process of transcription elongation
is continued by the core RNAP subunits. Elongating
RNAP sometimes associates with additional protein
factors known as elongation factors which can modulate
its properties. In general, elongation is highly processive:
the RNAP that begins an RNA chain continues synthesis
until the chain is terminated.
The process of transcription termination, like that of
initiation, is critical for the controlled expression of
genetic information. At the end of many transcription
units the DNA sequence encodes a G:C-rich stem loop
structure in the RNA chain followed by a short region
rich in uridine. This structure functions as a transcription
terminator and interacts with the core RNAP leading to
dissociation of the ternary complex and release of
the terminated RNA chain. In other cases, protein
factors such as the rho protein can bind to unstructured
regions of RNA and trigger transcription termination.
Once released by the termination reaction, the core
RNAP must rebind to a
s
-subunit to reform holoenzyme
and thereby complete the transcription cycle.
Since bacteria lack a nuclear membrane, the processes
of transcription and translation can be coupled: ribo-
somes can bind to the mRNA chain as it emerges from
RNAP and immediately initiate protein synthesis.
Indeed, along actively transcribed genes several RNAP
molecules may be simultaneously engaged in RNA chain
elongation with each RNA chain in turn bound by one
or more ribosomes engaged in protein synthesis.
Biochemical Properties
As expected for a critical component of the transcription
apparatus,
s
-factors have been highly conserved through
evolution. Indeed,
s
-factors from one bacterium can
function, at least in some cases, with the core RNAP from
distantly related species. In general, all
s
-factors share
certain defining properties. They all bind to the core
RNAP, apparently at a common binding site, to form
a holoenzyme. The presence of
s
-factor determines the
sequence of the promoters that can be bound by the
corresponding holoenzyme. The
s
-subunit, in those cases
that have been studied, also plays a role in the initiation of
the DNA-melting step that is an obligatory prelude to
transcription initiation. Often, although perhaps not
always,
s
is released from the ternary complex during the
process of RNA chain elongation.
Bacterial
s
-factors can be divided, based in part on
their protein sequences, into two families: the
s
70
and the
s
54
families. All bacteria contain at least one member of
the
s
70
family that is required for the transcription of
those genes essential for growth under virtually all
conditions (so-called “housekeeping” functions). This is
referred to as the primary (or class 1)
s
-factor. In
addition, most bacteria contain at least one (and as
many as 50 or more) alternative
s
-factors (Table I). These
factors typically control genes needed for specialized
functions that may only be expressed under particular
growth conditions. Examples include genes needed for
stress responses, motility, sporulation, uptake or trans-
port of specific nutrients, or antibiotic production.
Most alternative
s
-factors are structurally related to
the primary (class 1)
s
-factors and are therefore
considered to be members of the
s
70
family. Some
alternative
s
-factors (class 2) are very similar to the
primary
s
-factors but are, however, dispensable for
growth. One example is the E. coli
s
S
(RpoS)
protein that becomes active in stationary phase cells.
A much larger class of alternative
s
-factors are
those more distantly related in sequence to the primary
s
-factor, and often lacking one or more the conserved
regions characteristic of the class 1 and 2 proteins. These
alternative
s
-factors (classes 3, 4, and 5) control a
wide-range of physiological processes and are often only
42
SIGMA FACTORS