FIG. 2.

The effect of substitutions in σ³² and promoter DNA on runoff transcription from a PgroE-like promoter and its variants. Runoff transcription was carried out on a DNA fragment containing PgroE and RNAP holoenzyme containing WT σ³² or H107A σ³². (A) Runoff transcription from PgroE with WT and H107A. Bands were assigned as indicated. (B) Quantification of the effect of the H107A substitution on transcription from PgroE and the minor promoter. Each bar represents the ratio of transcription by RNAP containing H107A σ³² and WT σ³² for that particular promoter. (C) Promoters used in this work. Base identities in the −10 and −35 regions are shown in bold. The top line shows the sequence of the WT E. coli PgroE; the DNA template used, extending upstream to position −109 and downstream to position +390 in relation to the transcription start site also contained a minor σ³² promoter, shown in the second line (important differences with the PgroE [above] are indicated by underlining). The bottom four templates have a PgroE-like promoter, identical to PgroE through position −9, used in prior work (12, 27), as well as variants of this promoter (with base sequence differences underlined). Sequences downstream of −9, derived from the pQF50K vector (27), are shown in lowercase letters. (D) Runoff transcription assays. Pairwise comparisons of transcription by RNAP containing WT (lanes 2, 4, 6, and 8) and H107A (lanes 3, 5, 7, and 9) σ³² from the bottom four templates indicated in panel C. Shown is the gel analysis of the RNA products, labeled by the inclusion of [³²P]UTP in the reaction mixtures. The bands were assigned as indicated. (E) Normalized levels of radioactivity in RNA bands resulting from transcription by RNAP containing WT and H107A σ³² on the promoters shown in panel D above. For each bar, representing one promoter, transcription with WT RNAP is 100%. mut, mutant.