Award details

Initiation elongation pausing and termination by T7 RNA polymerase: A single molecule approach to the enzymology of transcription

Reference	BB/C507088/1
Principal Investigator / Supervisor	Dr Christoph Baumann
Co-Investigators / Co-Supervisors	Dr James Hoggett, Dr Justin Molloy
Institution	University of York
Department	Biology
Funding type	Research
Value (£)	228,770
Status	Completed
Type	Research Grant
Start date	21/03/2005
End date	20/09/2008
Duration	42 months

Abstract

This research programme will investigate the load-dependent kinetics of the complete transcription cycle using an optical tweezers-based, single molecule transcription assay that allows intermittent and processive mechanical interactions between an RNA polymerase and DNA to be measured in real-time. The single molecule transcription assay involves tethering a single DNA molecule containing a promoter, pseudo-gene and terminator between two micron-sized beads held in optical tweezers. By moving the DNA molecule past an RNA polymerase-coated surface immobilised bead using a waveform oscillation applied to the upstream bead, DNA protein interactions can be detected as a decoupling of the motion of the passive downstream bead from the driven upstream bead. This experimental design enables multiple enzymatic turnovers by an individual molecule to be observed. Using the single-subunit RNA polymerase from T7 bacteriophage as a model system, the research programme will investigate: (i) the dependence of initiation and promoter escape on ribonucleotide and pyrophosphate concentrations, using DNA templates containing either a double-stranded promoter or a single-stranded gapped promoter, (ii) the effect of a positive or negative load applied to the RNA polymerase on transcription initiation, pausing and termination, and (iii) the mechanistic details of promoter escape using T7 RNA polymerase mutants favouring either processive abortive initiation or processive elongation.

Summary

unavailable

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	Industrial Biotechnology, Microbiology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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