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Mol Cell. 2018 May 17;70(4):695-706.e5. doi: 10.1016/j.molcel.2018.04.018.

Correlating Transcription Initiation and Conformational Changes by a Single-Subunit RNA Polymerase with Near Base-Pair Resolution.

Author information

1
Department of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; Department of Chemistry, Chung-Ang University, Seoul 06974, Korea.
2
Department of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
3
Department of Biochemistry and Molecular Biology, Rutgers University, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.
4
Department of Biochemistry and Molecular Biology, Rutgers University, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA. Electronic address: patelss@rwjms.rutgers.edu.
5
Department of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; Howard Hughes Medical Institute, Baltimore, MD 21205, USA; Departments of Biophysics and Biophysical Chemistry, Biophysics, and Biomedical Engineering, Johns Hopkins University, MD 21205, USA. Electronic address: tjha@jhu.edu.

Abstract

We provide a comprehensive analysis of transcription in real time by T7 RNA Polymerase (RNAP) using single-molecule fluorescence resonance energy transfer by monitoring the entire life history of transcription initiation, including stepwise RNA synthesis with near base-pair resolution, abortive cycling, and transition into elongation. Kinetically branching pathways were observed for abortive initiation with an RNAP either recycling on the same promoter or exchanging with another RNAP from solution. We detected fast and slow populations of RNAP in their transition into elongation, consistent with the efficient and delayed promoter release, respectively, observed in ensemble studies. Real-time monitoring of abortive cycling using three-probe analysis showed that the initiation events are stochastically branched into productive and failed transcription. The abortive products are generated primarily from initiation events that fail to progress to elongation, and a majority of the productive events transit to elongation without making abortive products.

KEYWORDS:

FRET; T7 RNA polymerase; abortive RNA; closed complex; fluorescence; open complex; promoter DNA; single-molecule; transcription initiation; transition to elongation

PMID:
29775583
PMCID:
PMC5983381
[Available on 2019-05-17]
DOI:
10.1016/j.molcel.2018.04.018
[Indexed for MEDLINE]

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