Kinetics of RNA polymerase initiation and pausing at the lambda late gene promoter in vivo

We have measured the kinetics of transcription initiation and pausing by Escherichia coli RNA polymerase (RNAP) at the bacteriophage lambda late promoter, pR's, in growing cells. RNAP initiating transcription from pR' pauses after transcribing 16 or 17 nucleotides, and escape from this pause could in theory be the rate-limiting step in promoter function. We tested this hypothesis by analyzing pausing and non-pausing variants of both the pR' promoter segment and a more active mutant version of pR'; we measured reporter gene expression and used KMnO4 footprinting to measure directly occupancy of the promoter and pause sites in growing cells. We find that RNAP paused at +16/+17 does not limit expression of pR'. However, RNAP paused at +16/+17 does limit expression from the more active promoter by impeding formation of open complex. Therefore, the activity of the late gene regulatory protein Q to suppress the early pause, in addition to its antitermination activity, is unlikely to be important in phage gene expression.