NDLI: Promoter Binding, Initiation, and Elongation By Bacteriophage T7 RNA Polymerase A SINGLE-MOLECULE VIEW OF THE TRANSCRIPTION CYCLE

Content Provider	Journal of Biological Chemistry (JBC)
Author	Skinner, Gary M. Baumann, Christoph G. Quinn, Diana M. Molloy, Justin E. Hoggett, James G.
Abstract	A single-molecule transcription assay has been developed that allows, for the first time, the direct observation of promoter binding, initiation, and elongation by a single RNA polymerase (RNAP) molecule in real-time. To promote DNA binding and transcription initiation, a DNA molecule tethered between two optically trapped beads was held near a third immobile surface bead sparsely coated with RNAP. By driving the optical trap holding the upstream bead with a triangular oscillation while measuring the position of both trapped beads, we observed the onset of promoter binding, promoter escape (productive initiation), and processive elongation by individual RNAP molecules. After DNA template release, transcription re-initiation on the same DNA template is possible; thus, multiple enzymatic turnovers by an individual RNAP molecule can be observed. Using bacteriophage T7 RNAP, a commonly used RNAP paradigm, we observed the association and dissociation (koff= 2.9 s–1) of T7 RNAP and promoter DNA, the transition to the elongation mode (kfor = 0.36 s–1), and the processive synthesis (kpol = 43 nt s–1) and release of a gene-length RNA transcript (∼1200 nt). The transition from initiation to elongation is much longer than the mean lifetime of the binary T7 RNAP-promoter DNA complex (koff > kfor), identifying a rate-limiting step between promoter DNA binding and promoter escape.
Related Links	http://www.jbc.org/content/279/5/3239.abstract
Ending Page	3244
Starting Page	3239
Page Count	6
File Format	HTM / HTML PDF
ISSN	00219258
Journal	Journal of Biological Chemistry (JBC)
Issue Number	5
Volume Number	279
DOI	10.1074/jbc.M310471200
e-ISSN	1083351X
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2004-01-30
Access Restriction	Open
Subject Keyword	Genes: Structure and Regulation
Alternative Title	Promoter Binding, Initiation, and Elongation By Bacteriophage T7 RNA Polymerase
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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Transient State Kinetics of Transcription Elongation by T7 RNA Polymerase

Binding of the Priming Nucleotide in the Initiation of Transcription by T7 RNA Polymerase

T7 Lysozyme Represses T7 RNA Polymerase Transcription by Destabilizing the Open Complex during Initiation

Multiple Roles for the T7 Promoter Nontemplate Strand during Transcription Initiation and Polymerase Release

Kinetic Mechanism of GTP Binding and RNA Synthesis during Transcription Initiation by Bacteriophage T7 RNA Polymerase

Cross-linking of Promoter DNA to T7 RNA Polymerase Does Not Prevent Formation of a Stable Elongation Complex

Inhibition of TATA Binding Protein Dimerization by RNA Polymerase III Transcription Initiation Factor Brf1

Observed Instability of T7 RNA Polymerase Elongation Complexes Can Be Dominated by Collision-induced “Bumping”

Bacteriophage T7 transcription system: an enabling tool in synthetic biology

Promoter Binding, Initiation, and Elongation By Bacteriophage T7 RNA Polymerase A SINGLE-MOLECULE VIEW OF THE TRANSCRIPTION CYCLE

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Promoter Binding, Initiation, and Elongation By Bacteriophage T7 RNA Polymerase A SINGLE-MOLECULE VIEW OF THE TRANSCRIPTION CYCLE