Bacterial transcription has been studied extensively in vitro, which has provided detailed molecular mechanisms of transcription. The in vivo cellular environment, however, may impose different rules on transcription than the homogeneous and well-controlled in vitro environment. How an RNA polymerase (RNAP) molecule searches rapidly through vast nonspecific chromosomal DNA in the three-dimensional nucleoid space and identifies a specific promoter sequence remains elusive. Transcription kinetics in vivo could also be impacted by specific cellular environments including nucleoid organization and nutrient availability. In this work, we investigated the promoter search dynamics and transcription kinetics of RNAP in live E. coli cells. Using single-molecule tracking (SMT) and fluorescence recovery after photobleaching (FRAP) across different genetic, drug inhibition, and growth conditions, we observed that RNAP’s promoter search is facilitated by nonspecific DNA interactions and is largely independent of nucleoid organization, growth condition, transcription activity, or promoter class. RNAP’s transcription kinetics, however, are sensitive to these conditions and mainly modulated at the levels of actively engaged RNAP and the promoter escape rate. Our work establishes a foundation for further mechanistic studies of bacterial transcription in live cells.

Bacterial transcription has been studied extensively in vitro, which has provided in-depth insight regarding transcription mechanisms. However, the live cell environment may impose different rules on transcription than the homogenous and simplified in vitro environment. How an RNA polymerase (RNAP) molecule searches rapidly through the vast nonspecific chromosomal DNA in the three-dimensional nucleoid space and binds a specific promoter sequence remains elusive. The kinetics of transcription in vivo could also be impacted by specific cellular environments including nucleoid organization and nutrient availability. In this work, we investigated the promoter search dynamics and transcription kinetics of RNAP in live E. coli cells. Using single-molecule tracking (SMT) and fluorescence recovery after photobleaching (FRAP) and combining with different genetic, drug inhibition, and growth conditions, we observed that the promoter search of RNAP is facilitated by nonspecific DNA interactions and largely independent of nucleoid organization, growth condition, transcription activity, or promoter classes. The transcription kinetics of RNAP, however, is sensitive to these conditions and mainly modulated at the levels of actively engaged RNAP and the promoter escape rate. Our work establishes a foundation for further mechanistic studies of bacterial transcription in live cells.