RNA Polymerase Pausing during Initial Transcription.
In bacteria, RNA polymerase (RNAP) initiates transcription by synthesizing short transcripts that are either released or extended to allow RNAP to escape from the promoter. The mechanism of initial transcription is unclear due to the presence of transient intermediates and molecular heterogeneity. Here, we studied initial transcription on a lac promoter using single-molecule fluorescence observations of DNA scrunching on immobilized transcription complexes. Our work revealed a long pause ("initiation pause," ∼20 s) after synthesis of a 6-mer RNA; such pauses can serve as regulatory checkpoints. Region sigma 3.2, which contains a loop blocking the RNA exit channel, was a major pausing determinant. We also obtained evidence for RNA backtracking during abortive initial transcription and for additional pausing prior to escape. We summarized our work in a model for initial transcription, in which pausing is controlled by a complex set of determinants that modulate the transition from a 6- to a 7-nt RNA.
Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, UK.
Diego
D
Duchi
Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, UK.
David L V
DL
Bauer
Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, UK.
Laurent
L
Fernandez
Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, UK.
Geraint
G
Evans
Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, UK.
Nicole
N
Robb
Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, UK.
Ling Chin
LC
Hwang
Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, UK.
Kristofer
K
Gryte
Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, UK.
Alexandra
A
Tomescu
Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, UK.
Pawel
P
Zawadzki
CNRS FRE 3689, Centre d'études d'agents Pathogénes et Biotechnologies pour la Santé (CPBS), 1919 route de Mende, 34293 Montpellier, France.
Zakia
Z
Morichaud
CNRS FRE 3689, Centre d'études d'agents Pathogénes et Biotechnologies pour la Santé (CPBS), 1919 route de Mende, 34293 Montpellier, France.
Konstantin
K
Brodolin
Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, UK. Electronic address: kapanidis@physics.ox.ac.uk.
Achillefs N
AN
Kapanidis
D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D007763:Lac Operon; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D012293:Rifampin / Q000494:pharmacology; D000072760:Single Molecule Imaging / Q000379:methods; D013997:Time Factors; D014158:Transcription, Genetic
D029968:Escherichia coli Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin
DNA scrunching; RNA polymerase; initial transcription; promoter escape; single-molecule FRET; transcriptional pausing