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

DISCUSSION
4.3.2 Mechanism of transcriptional activation of the tyrT promoter
Stable RNA promoters in E. coli are both highly regulated and show a high maximal
rate of transcription initiation. The tyrT UAS is necessary for optimal expression in vivo
[Lamond & Travers, 1983] and can activate transcription in both FIS-dependent and
independent modes. In vitro the tyrT UAS increases the affinity of the polymerase for
the promoter [Sander et al., 1993] and has been inferred to make a contact with RNA
polymerase at positions around -120 to -130 on a negatively supercoiled DNA template
[Pemberton et al., 2002]. We note that using conditions where we would expect this
contact to be formed efficiently we do not observe untwisting of the -10 region in the
wild-type promoter (Figure 21A), although the -10 hexamer is contacted prior to the
formation of the upstream contact [Pemberton et al., 2002]. However, untwisting does
occur in the presence of FIS (Figure 21A). This is consistent with both previous
observation that FIS facilitates DNA untwisting in the -10 region in the presence of the
initiating NTPs [Muskhelishvili et al., 1997] and also with other observations that FIS
can rescue the impaired activity of a promoter with down mutation in the -10 region
[Lazarus & Travers, 1993] and of a mutant RNA polymerase whose binding to the rrnB
P1 promoter is destabilized [Bartlett et al., 2000]. The finding that the -35U and spacer
mutants simultaneously lose the dependence on negative superhelicity and the
requirement for the UAS in vivo suggests that these mutations overcome wholly or
partially the barrier imposed by the wild-type promoter organization and hence abolish
the requirement for activation by the UAS alone or by FIS bound to the UAS. The UAS
thus acts primarily as a regulatory region with a distinct DNA geometry that is sensitive
to the superhelical density of the DNA template [Travers & Muskhelishvili, 1998].
Stabilization of this geometry by FIS buffers polymerase function against variations in
negative superhelicity. Provision of such a buffer by FIS operates both for the tyrT
promoter (this work) and also for the rrnA P1 promoter [Rochman et al., 2002]. We
would expect this mechanism to be common to all promoters of similar organization.
From the data here we infer that the regulated step in transcription initiation at the tyrT
and possibly other stable RNA promoters is the untwisting of the -10 region, a step
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