(Converted)-5 - Journal of Cell and Molecular Biology - Haliç ...

68 Nihal Büyükuslu
spectroscopy showed that the structure of αCTD is
compactly folded and comprised of four helices and
two long loops at the ends of the domain. A chemical
shift perturbation experiment was performed to
observe which residues of αCTD are involved in the
interaction with UP promoter elements. The residues
affected by perturbation were attributable to amides of
most of the residues from Glu261 to Ile275 and from
Thr292 to Ile303 that are located in helix 1, the Nterminal
end of the helix 4, and the preceding loop.
The C-terminal region of the α subunit is
responsible for contact with cis-acting UP element as
well as with trans-acting transcription factors. One of
the well characterized transcription factors, cAMP-
CRP, binds to specific (22bp) sites on DNA, the
position of the site(s) depending on the particular
promoters. The cAMP receptor protein, CRP, controls
the initiation of transcription of several genes
especially those involved in carbon-source utilization.
Activation of transcription by the upstream CRP
molecule is blocked by the HL159 substitution,
suggesting that the upstream-bound CRP makes a
direct contact with RNA polymerase. Footprinting
experiments indicated that RNA polymerase contacts
the promoter DNA between the two CRP-binding sites,
most likely due to interactions involving the C-terminal
part of the α subunit (Attey et al., 1994). Although
many CRP-dependent promoters carry a single CRPbinding
site, centered around –40, –60 or –70, a
number of promoters carry multiple CRP-binding sites.
In order to accommodate direct contacts between both
CRP dimers and the two α subunits in ternary
complexes at the ML1 promoter, Busby et al. (1994)
proposed a model that the α subunits are sandwiched
between CRP and RNA polymerase. Since the α dimer
is able to bind directly to DNA (Ross et al., 1993) it
seems probable that the α subunits are responsible for
the upstream RNA polymerase contacts and that the α
dimer bridges the two CRP dimers. In contrast, at class
II promoters, α binds just upstream of the CRP dimer
and makes contact with via activating region I.
Although the CRP dimer contains an activating region
I in each subunit, it is only the activating region I in the
upstream of the CRP dimer that makes contact with
RNA polymerase during transcription initiation. These
results suggest that α makes contact with the upstream
subunit of the CRP dimer whilst the downstream
subunit is likely to make alternative contacts with other
parts of RNA polymerase (Attey et al., 1994).
The ß subunit
The second largest subunit of E. coli RNA polymerase
is composed of 1342 amino acids and is highly
conserved throughout evolution (Sweetser et al., 1987;
Iwabe et al., 1991; Ovchinnikov et al., 1981). The β
subunit alone has no apparent function like the other
subunits. When assembled into the RNA polymerase
complex, β subunit has been shown to be involved in
most of the catalytic functions of RNA polymerase,
including nucleotide binding (Jin and Gross, 1991;
Mustaev et al., 1991), transcription initiation,
elongation and termination (Mustaev et al., 1991; Jin
and Gross, 1988; Kashlev et al., 1990; Landick et al.,
1990; Lee and Goldfarb 1991; Jin 1994), interactions
with both the σ subunit (Glass et al., 1986;1988) and
the NusA proteins (Jin and Gross, 1988; Sparkowski
and Das, 1992). Mutations conferring resistance to
rifampicin define several clustered residues in the
central and amino terminal parts of the β subunit
(Lisitsyn et al., 1984; Severinov et al., 1993;
Ovchinnikov et al., 1981; 1983; Jin and Gross 1988).
Sequence similarities among the subunits of
different organisms implied three main domains; Nterminal
domain, middle domain and C-terminal
domain, and two dispensable regions centered around
residues 300 and 1000. The conserved regions are
thought to be important for function and structure of β
subunit and the homology of these regions among
organisms reveal the evolution of genetic information
flow.
Deletions in the N-terminal region of subunit of E.
coli RNA polymerase between the residues 166-328
and 186-433 showed no obvious effect on function in
vitro, suggesting that this region is dispensable for
minimal function. The ∆(166-328) alteration was also
found to be non-lethal in vivo (Severinov et al., 1994).
A 69-residue segment between the residues 339-409 of
β subunit is widespread among prokaryotes indicating
that this region might play a structural or functional
role. Indeed, a recent study showed that the β subunit
residues 186-433 and 436-445 are commonly used by
σ 54 and σ 70 RNA polymerase holoenzyme for open
promoter complex formation (Wigneshweraraj et al.,
2002). In E. coli this region also contains the paf32
alteration (Severinov et al., 1994) due to a contact site
with the Alc protein, a site-specific termination factor
encoded by bacteriophage T4 that acts as a block to the
transcription of host genes (Kashlev et al., 1993). In
support of this, Landick et al. (1990) identified a series