Abstract Bacterial transcription has been studied extensively in vitro , which has provided indepth 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 RNAP’s promoter search is facilitated by nonspecific DNA interactions and largely independent of nucleoid organization, growth condition, transcription activity, or promoter classes. RNAP’s transcription kinetics, 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.

RNAP promoter search and transcription kinetics in liveE. colicells