Function of E. coli RNA Polymerase σ Factor- σ70 in Promoter-Proximal Pausing

conformation or components to be modified in vitro by the gene Q antiterminator, as assayed either by foot-printing of Q protein bound in the complex or by transcription antitermination downstream; a complex made Summary of mutant DNA (e.g., mutant at ϩ2 or ϩ6), in which RNA synthesis is stopped by lack of a nucleotide substrate at The ␴ factor ␴ 70 of E. coli RNA polymerase acts not only the site of the wild-type pause, is competent for neither in initiation, but also at an early stage of elongation to modification (Yarnell and Roberts, 1992). induce a transcription pause, and simultaneously to The transcribing complex paused at ϩ16/17 has many allow the phage ␭ gene Q transcription antiterminator essential properties of an elongation complex. Ac-to act. We identify the signal in DNA that induces early cording to both MPE and exonuclease footprinting, the pausing to be a version of the ␴ 70 Ϫ10 promoter con-RNA polymerase-␴ 70 contact has been released from sensus, and we show that ␴ 70 is both necessary for the Ϫ35 segment, and, almost certainly, from the Ϫ10 pausing and present in the paused transcription com-segment as well, although some pause-specific interac-plex. Regions 2 and 3 of ␴ 70 suffice to induce pausing. tions do occur near the Ϫ10 segment (Yarnell and Rob-Since pausing is induced by the nontemplate DNA erts, 1992). The length of the exonuclease footprint, strand of the open transcription bubble, we conclude about 30–35 nucleotides, is typical of elongation com-that RNA polymerase containing ␴ 70 carries out base-plexes. The paused complex is stable to incubation and specific recognition of the nontemplate strand as sin-to filtration, in contrast to complexes that contain abor-gle stranded DNA. We suggest that ␴ 70 remains bound tive RNAs. RNA polymerase initiated at the late gene to core RNA polymerase when the Ϫ10 promoter con-promoter of a related lambdoid phage, phage 82, contacts are broken, and then moves to the pause-induc-tains RNA paused at ϩ25, even farther from the pro-ing sequence. These characteristics would give rise to the expecta-Introduction tion that ␴ 70 has been released at the ϩ16/17 pause. In contrast, we have found that ␴ 70 is present in the paused ␴ factors of bacterial RNA polymerase mediate recogni-complex—both at ϩ16/17 for ␭ and at ϩ25 for phage tion of promoters, and possibly the opening of duplex 82—and that ␴ 70 is necessary for the pause …