Mutations in the NPDKV and DGGGR alter the structural features
of the promoter recognition sites
Structural superimposition of ๐2 and
๐4 domains in RpoE10 with other ๐/anti- ๐ complexes31,32 revealed that in RpoE10, orientation and
accessibility of DNA binding surfaces are exposed (Figure 4A). Like its
template Mtb-SigJ 18, RpoE10 lacks the first helix ฮฑ1,
usually present in the ฯ2 domain, which has three
helices ฮฑ2โฮฑ3- ฮฑ4 connected by two loops L2 and L3. Intriguingly, a
comparative analysis of the average fluctuations of all the backbone
atoms of the amino acid residues (RMSF profile) of the RpoE10, RpoE10
(Mut1), and RpoE10 (Mut2) showed a contrasting pattern at key positions
(Figure 4A). The ฮฑ1-helix showed an increased RMSF fluctuation in RpoE10
compared to both RpoE10 (Mut1 and Mut2) (marked as 1 in Figures 4A and
B). Remarkably, we noticed a prominent fluctuation in the L3 loop
(residues 46-52) between second and third helices (ฮฑ2 andฮฑ3) of RpoE10
(Mut1) (marked as 2 in Figure 4A and B). It has been shown that the
flexible โspecificity loopโ initiates promoter recognition and
determines ECF ฯ factorsโ specificity at the โ10 promoter element in ECF
ฯ-dependent promoters 32. Therefore, a reduced
fluctuation in the ฮฑ1 helix and a simultaneously increased fluctuation
in the L3 loop of the ๐2 domain could favor
recognizing -10 promoter and DNA melting by RpoE10 (Mut1), leading to
enhanced promoter activation.
Also, in RpoE10, increased fluctuation at the linker-loop junction
connecting the ๐2 and ๐4 domains
(peaks are marked as 3 and 4 in Figure 4B), suggests the possibility of
conformational instability of ๐2 and
๐4 domains. As compared to RpoE10(Mut2), the junction
region (marked as 4 in Figure 4B) towards the ๐4domain
is stabilized in RpoE10 (Mut1). Therefore, similar to Mtb-SigJ (18), the
decreased fluctuations at the junction of
๐2-linker-๐4 region depicts the
stabilized and tethered ๐2 and ๐4domains essential for acquiring a productive conformation of
RpoE10(Mut1) for enhanced activation of its promoter. Next, we focused
on another important segment (135-151)of the ฯ4domain, a helix-turn-helix motif known to interact with the โ35 element
of the promoter 34. Both, RpoE10 and RpoE10(Mut2)
showed an enhanced average fluctuation of backbone atoms of residues
135-151 segment as compared to RpoE10(Mut1) (peaks are marked as 5 in
Figure 4 A and B). Unlike RpoE10 and RpoE10(Mut2), the stabilized
helix-turn-helix motif of ฯ4 domain of RpoE10(Mut1)
reasonably favors interaction with the โ35 element of the promoter and,
therefore, justifies its enhanced activation.
Interestingly, we notice that the RMSF profile of the RpoE10 (Mut1 and
Mut2) showed increased conformational flexibility at both NPDKVand DGGGR
motifs (marked as 6 and 9 respectively, in Figure 4B), as compared to
that in RpoE10. Since the DGGGR motif is essential for promoter
activation (Figure 1), the stabilized conformation of backbone atoms of
the NPDKV motif in RpoE10 raises the possibility to obstruct the DGGGR
motif away from the ๐2-๐4 linker
site. In this situation, the stabilized orientation of the NPDKV motif
may lead to the formation of unproductive conformations of2-๐4 domain in RpoE10, and therefore
a possible reason for the elimination of its activity. Notably, in
RpoE10, another segment of 230-238 residues (marked as 8 in Figures 4A
and B) showed a sharp and distinct rise in the fluctuations of backbone
atoms compared to RpoE10(Mut1 and Mut2). This segment forms the core of
the SnoaL_2 domain, and therefore the backbone fluctuations may act as
a trigger signal for eliminating the promoter activation of RpoE10.
RMSF analysis showed that the NPDKV mutant form of RpoE10 containing an
intact DGGGR motif showed stable conformations at the -35 promoter
binding site (residues 135-151), enhanced flexibility of L3
โspecificity loopโ (residues 45-52) around the -10 promoter
recognition site, and stabilized linker region connecting the
๐2-๐4 domain which are essential
features to attain a productive conformation for enhanced promoter
activation.