Real-time observation of the transition from transcription initiation to elongation of the RNA polymerase.

Abstract

The transition from initiation to elongation of the RNA polymerase (RNAP) is an important stage of transcription that often limits the production of the full-length RNA. Little is known about the RNAP transition kinetics and the steps that dictate the transition rate, because of the challenge in monitoring subpopulations of the transient and heterogeneous transcribing complexes in rapid and real time. Here, we have dissected the complete transcription initiation pathway of T7 RNAP by using kinetic modeling of RNA synthesis and by determining the initiation (IC) to elongation (EC) transition kinetics at each RNA polymerization step using single-molecule and stopped-flow FRET methods. We show that the conversion of IC to EC in T7 RNAP consensus promoter occurs only after 8- to 12-nt synthesis, and the 12-nt synthesis represents a critical juncture in the transcriptional initiation pathway when EC formation is most efficient. We show that the slow steps of transcription initiation, including DNA scrunching/RNAP-promoter rotational changes during 5- to 8-nt synthesis, not the major conformational changes, dictate the overall rate of EC formation in T7 RNAP and represent key steps that regulate the synthesis of full-length RNA.