Fig. 7.

Effect of substitution at K130 of σ³² on promoter recognition in vitro. Single-round in vitro transcriptions were performed on linear DNA templates containing wt or −16,−15 AA mutant groE promoters by RNA polymerase containing E. coli wt σ³², K130A σ³² or C. crescentus s ³²(CCRσ³²).

A. In vitro transcription from groE promoter variants with σ³² variants. Upper bands in each lane are from long template of wt groE promoter (served as internal control); lower bands are from short template of either wt or −16,−15 AA mutant groE promoter (see Experimental procedures); bottom bands (end-labelled 35 nt oligomer) are shown as loading control.

B. Quantification of the in vitro transcripts to show the effect of substitution at K130 position on promoter recognition. The bars indicate the relative transcription from −16, −15 AA mutant promoter as a percentage transcript from wt promoter for each σ³² variant. Each value was calculated as follows: (i) each short transcript was expressed as a fraction of total transcript in the lane (long + short transcript) and called normalized short transcript and (ii) normalized short transcript from −16, −15 AA promoter was divided by normalized short transcript from the wt promoter. All values are averages of three independent experiments; error bars indicate one standard deviation.