RT Journal Article SR Electronic T1 A Novel Initiation Pathway in Escherichia Coli Transcription JF bioRxiv FD Cold Spring Harbor Laboratory SP 042432 DO 10.1101/042432 A1 Eitan Lerner A1 SangYoon Chung A1 Benjamin L. Allen A1 Shuang Wang A1 Jookyung J. Lee A1 Winson S. Lu A1 Wilson L. Grimaud A1 Antonino Ingargiola A1 Xavier Michalet A1 Yazan Alhadid A1 Sergei Borukhov A1 Terence Strick A1 Dylan J. Taatjes A1 Shimon Weiss YR 2016 UL http://biorxiv.org/content/early/2016/03/27/042432.abstract AB Initiation is a highly regulated, rate-limiting step in transcription. We employed a series of approaches to examine the kinetics of RNA polymerase (RNAP) transcription initiation in greater detail. Quenched kinetics assays, in combination with magnetic tweezer experiments and other methods, showed that, contrary to expectations, RNAP exit kinetics from later stages of initiation (e.g. from a 7-base transcript) was markedly slower than from earlier stages. Further examination implicated a previously unidentified intermediate in which RNAP adopted a long-lived backtracked state during initiation. In agreement, the RNAP-GreA endonuclease accelerated transcription kinetics from otherwise delayed initiation states and prevented RNAP backtracking. Our results indicate a previously uncharacterized RNAP initiation state that could be exploited for therapeutic purposes and may reflect a conserved intermediate among paused, initiating eukaryotic enzymes.Significance: Transcription initiation by RNAP is rate limiting owing to many factors, including a newly discovered slow initiation pathway characterized by RNA backtracking and pausing. This backtracked and paused state occurs when all NTPs are present in equal amounts, but becomes more prevalent with NTP shortage, which mimics cellular stress conditions. Pausing and backtracking in initiation may play an important role in transcriptional regulation, and similar backtracked states may contribute to pausing among eukaryotic RNA polymerase II enzymes.