Coordinated regulation of gene expression by E ... - Jacobs University
Coordinated regulation of gene expression by E ... - Jacobs University
Coordinated regulation of gene expression by E ... - Jacobs University
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RESULTS<br />
Figure 19: In vitro transcription <strong>of</strong> ∆61 constructs <strong>of</strong> wild type and derivatives.<br />
In vitro transcription assay was performed from supercoiled (σ = -0.075) and linearized templates.<br />
Transcripts originating from bla <strong>gene</strong> on the same plasmid is also shown. SC – supercoiled templates; lin<br />
– linear templates.<br />
The tyrT wild-type and tyrTD promoter constructs, which clearly showed<br />
sensitivity to supercoiling, were investigated in more detail to elucidate the extent <strong>of</strong><br />
activation <strong>by</strong> FIS at different superhelical densities. The activity <strong>of</strong> the wild-type<br />
promoter showed a more strict dependence on supercoiling level for its activity, while<br />
tyrTD was active under a broader range <strong>of</strong> superhelical densities (Figure 20A). Presence<br />
<strong>of</strong> FIS was seen to activate the transcription at all supercoiling levels in both wild-type<br />
and the discriminator mutant, but the activation <strong>of</strong> the wild-type was more significant<br />
under non-optimal supercoiling levels (σ = - 0.036 & -0.068) (Figure 20B).<br />
3.2.5 Effects <strong>of</strong> promoter mutations on the function<br />
Since the data obtained implicated the mutations in the core promoter region in the<br />
altered sensitivity to both supercoiling and FIS we next investigated the effects <strong>of</strong> these<br />
mutations on promoter function <strong>by</strong> using potassium permanganate footprinting<br />
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