Tethering sigma70 to RNA polymerase reveals high in vivo activity of sigma factors and sigma70-dependent pausing at promoter-distal locations.
Bacterial sigma factors compete for binding to RNA polymerase (RNAP) to control promoter selection, and in some cases interact with RNAP to regulate at least the early stages of transcript elongation. However, the effective concentration of sigmas in vivo, and the extent to which sigma can regulate transcript elongation generally, are unknown. We report that tethering sigma70 to all RNAP molecules via genetic fusion of rpoD to rpoC (encoding sigma70 and RNAP's beta' subunit, respectively) yields viable Escherichia coli strains in which alternative sigma-factor function is not impaired. beta'::sigma70 RNAP transcribed DNA normally in vitro, but allowed sigma70-dependent pausing at extended -10-like sequences anywhere in a transcriptional unit. Based on measurement of the effective concentration of tethered sigma70, we conclude that the effective concentration of sigma70 in E. coli (i.e., its thermodynamic activity) is close to its bulk concentration. At this level, sigma70 would be a bona fide elongation factor able to direct transcriptional pausing even after its release from RNAP during promoter escape.
Mooney
Rachel Anne
RA
Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Landick
Robert
R
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D015964:Gene Expression Regulation, Bacterial; D006358:Hot Temperature; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012808:Sigma Factor / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012808:Sigma Factor; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases