Real-Time Transcription Initiation by E. coli RNA Polymerase in vitro and in vivo

Abstract

Transcription initiation is the most important step in gene regulation and is orchestrated by RNA polymerase (RNAP). However, its molecular mechanisms have remained unclear due to the presence of transient intermediates and heterogeneity.To characterize such mechanisms, we used an in vitro real-time FRET assay on immobilized transcription complexes for minute-long observations of DNA scrunching and unscrunching. We characterized the kinetics of abortive initiation (by following single cycles of abortive synthesis) and promoter escape, and identified functionally important heterogeneity. We observed, for the first time, extensive RNAP pausing (lasting for ∼15 s) and backtracking during initiation; such behaviors may play regulatory roles.We also studied initial transcription in vivo using electroporation to internalize doubly-labeled promoter DNA fragments into live E. coli and track them using TIRF microscopy. We observed low-FRET species of 0.18±0.05 (corresponding to duplex DNA) and fluctuations to higher FRET-states, which we attribute to RNAP promoter binding, open complex formation and initial transcription. Specifically, we see FRET of 0.35±0.10 (linked to initiation pausing), and 0.85±0.06 (linked to promoter escape); both levels are absent in non-promoter DNA.Our work reveals the detailed kinetics of initial transcription in vitro and offers the first such observations in living cells, which opens exciting avenues to study gene regulation in vivo.