In vitro transcription/primer extension of gyrB . In vitro transcription, followed by primer extension, was performed on the gyrB promoter template pJEKS-P3. A σ70 RNAP holoenzyme preparation, obtained from E. coli σ70 was used for transcription assays. Shown at the top are the DNA species, produced from the extended messages, next to a sequencing ladder generated from the same primer. Strong start sites are indicated by larger arrows, weaker start sites by smaller arrows. Below, is the complimentary sequence of the gyrB promoter, with the -10 and -35 promoter elements indicated. The major transcription start sites found by this assay are shown by upward-facing arrows, while the adjacent start sites previously found by primer extension (see Fig. 1) are shown in bold; the largest upward-facing arrow indicates a start site appearing in both assays and with both RNA polymerase species.