Coordinated regulation of gene expression by E ... - Jacobs University

DISCUSSION
writhe in the UAS by FIS protects rrnA P1 promoters from inactivation at sub-optimal
superhelical densities. This compensatory effect is also apparent with wild-type tyrT in
vivo when expressed from hyperhegatively supercoiled plasmid DNA. We see here that
the mutations in the core promoter that alter the sensitivity to supercoiling also alter the
response to FIS, although the UAS region containing the FIS binding sites remain
intact. Also the increased supercoiling of the wild-type promoter template in the fis cells
[Auner et al., 2003] may, in principle compensate for the absence of FIS. The distinct
roles of core promoter and UAS in sensing the supercoiling and FIS revealed in this
work, together describe the extent of regulatability built into the stringently controlled
tyrT promoter.
4.3.1 Promoter structure and function
We have studied the effects of the three mutations of tyrT core promoter: a change in
the -35 region to the consensus sequence TTGACA, an increase in the spacing between
the –10 and –35 hexamers from a suboptimal 16bp to the consensus 17bp [Lisser et al.,
1993], and a 4bp substitution in the discriminator region (TTAA in place of CCCC,
Figure 16B). The mutations towards the consensus exhibit substantially increased
activity in vivo (Figure 17).
The permanganate reactivity of promoter mutants’ show that all the core
promoter mutants studied in the absence of FIS and NTPs have enhanced interactions
with polymerase relative to the wild-type promoter in the TG of the extended –10
region and the –10 region itself (Figure 21A). The mutation in the spacer and –35
hexamer also result in the loss of dependence on negative superhelicity, showing that
the sensitivity of the tyrT promoter to this parameter is determined by more than one
aspect of sequence organization. A further similarity is that both these mutations act in
vitro to enhance interactions with the –10 region (Figure 21). Notably, neither of these
mutations enhanced permanganate reactivity in the vicinity of the transcription start
point in the absence of NTPs. We infer that a major effect of these mutations is to
promote untwisting of DNA within the –10 hexamer. The inference we draw is that a
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