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

RESULTS
3.2 Role of supercoiling and chromatin protein FIS in
transcriptional regulation of an individual promoter
The current section describes the role of supercoiling and FIS in activation of the
promoter of one of the stable RNA promoters, tyrT. The obtained results give an insight
into the mechanistic basis of interplay of these regulators at the molecular level.
3.2.1 Mechanism of transcriptional regulation by FIS &
supercoiling: tyrT promoter paradigm
It is known that the tyrT and other stable RNA promoters are regulated by FIS and
negative supercoiling [Lazarus & Travers, 1993; Muskhelishvili et al., 1995 and 1997;
Free & Dorman, 1994; Bowater et al., 1994]. Figure 16A shows the sequence
organization of tyrT promoter. To address the role of the core promoter structure in the
response of the tyrT promoter to activation by FIS and regulation by supercoiling, three
different “up” mutations were introduced in the tyrT core promoter region: a -35 “up”
mutation (35U), a discriminator mutation (D), and an A-T base-pair insertion at position
-22 in the spacer region between the -10 and -35 elements (S) (Figure 16B). The
mutations in the -35 region and in the spacer (35U and S) increase the homology of the
promoter to the consensus polymerase binding sequence and the discriminator mutation
(D) relieves the block imposed by the G/C-rich discriminator. Both the wild-type and
truncated promoter constructs lacking sequences beyond position -61 and thus devoid of
UAS with all three FIS binding sites were used for the study. These latter promoter
constructs are designated ∆61 promoters.
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