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

DISCUSSION
primary effect of negative superhelicity on the tyrT promoter is to drive the untwisting
of the –10 region.
The discriminator region has previously been implicated as an important
determinant of transcriptional response to negative supercoiling at hisR and tyrT
promoters [Figueroa-Bossi et al., 1998; Pemberton et al., 2000]. In the experiments
described here we have shown that in the absence of the initiating NTPs a discriminator
mutation enhances contacts around – 10 region and the transcription start point,
implying that the mutation relieves a discriminator-dependent block on open complex
formation. Also, the tyrTD mutation reduces the optimum superhelical density for
transcription in vitro. However, in the LZ strains the discriminator mutant is still
strongly activated by negative superhelicity and is less active at hypernegative sigma
values (Figure 18). Taken together these observations suggest that an intact
discriminator enhances the response of a promoter to a negative superhelicity and that
overall its mutation attenuates, but does not abolish, the response.
We found that mutation of the –35 region or the spacer region to the consensus
abolishes dependence of expression on negative superhelicity in vivo while mutation of
the discriminator does not. We infer that the response to superhelicity is modulated by
the primary promoter elements (-35 hexamer, spacer, -10 hexamer). These elements
individually or together, determine the torque available for nucleation, the polymerasedependent
untwisting of the –10 region. The role of the discriminator element is
different. This element blocks the subsequent propagation of untwisting from the –10
region to the transcription start point and hence constitutes a barrier to open complex
formation. Even when nucleation was promoted by mutation of the spacer or the –35
region no further progression of the untwisted region to the start point was observed
(Figure 21A) presumably because these variant promoters still contained an intact
discriminator. One explanation for the partial alleviation of the response to supercoiling
by the tyrTD mutation would be lowering of the activation energy for untwisting of the
–10 region by virtue of its proximity [Drew et al., 1985].
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