Monitoring RNA transcription in real time by using surface plasmon resonance.
The decision to elongate or terminate the RNA chain at specific DNA template positions during transcription is kinetically regulated, but the methods used to measure the rates of these processes have not been sufficiently quantitative to permit detailed mechanistic analysis of the steps involved. Here, we use surface plasmon resonance (SPR) technology to monitor RNA transcription by Escherichia coli RNA polymerase (RNAP) in solution and in real time. We show that binding of RNAP to immobilized DNA templates to form active initiation or elongation complexes can be resolved and monitored by this method, and that changes during transcription that involve the gain or loss of bound mass, including the release of the sigma factor during the initiation-elongation transition, the synthesis of the RNA transcript, and the release of core RNAP and nascent RNA at intrinsic terminators, can all be observed. The SPR method also permits the discrimination of released termination products from paused and other intermediate complexes at terminators. We have used this approach to show that the rate constant for transcript release at intrinsic terminators tR2 and tR' is approximately 2-3 s(-1) and that the extent of release at these terminators is consistent with known termination efficiencies. Simulation techniques have been used to fit the measured parameters to a simple kinetic model of transcription and the implications of these results for transcriptional regulation are discussed.
Greive
Sandra J
SJ
Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, OR 97403, USA.
Weitzel
Steven E
SE
Goodarzi
Jim P
JP
Main
Lisa J
LJ
Pasman
Zvi
Z
von Hippel
Peter H
PH
D012321:DNA-Directed RNA Polymerases; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D012329:RNA, Bacterial / Q000096:biosynthesis*; D012808:Sigma Factor; D020349:Surface Plasmon Resonance / Q000379:methods*; D013728:Terminator Regions, Genetic; D024363:Transcription Initiation Site; D014158:Transcription, Genetic
D012329:RNA, Bacterial; D012808:Sigma Factor; D012321:DNA-Directed RNA Polymerases