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Transcription: Structural Basis of Promoter Escape


EMSL Project ID
50695

Abstract

We propose to define the structural and mechanistic basis of promoter escape in bacterial transcription initiation by use of single-particle-reconstruction cryo-EM to determine structures of a set of bacterial transcription initiation complexes containing RNA products of lengths 11, 12, 13, 14, and 15 nucleotides. The results will provide a foundation for understanding bacterial promoter escape and will be relevant to understanding transcription initiation in all organisms, from bacteria through eukaryotes.
In preliminary work, we have developed and validated procedures to prepare homogenous functional recombinant transcription complexes that contain RNAP sigmaA holoenzyme from the hyperthermophilic bacterium T. thermophilus and that contain RNA products of lengths 11, 12, 13, 14, and 15 nucleotides.
In the proposed work, we will screen samples of each of the five transcription complexes on a Talos Arctica (one day per complex; five days total), and, for samples yielding satisfactory ice thicknesses, particle distributions, and particle orientations, we will process data to obtain initial reconstructions and will attempt to fit initial reconstructions using crystal structures of RNAP sigmaA holoenzyme, transcription initiation complexes, and transcription elongation complexes. We will use the resulting initial reconstructions and provisional fitted pseudoatomic models (i) to generate initial hypotheses and (ii) to apply for access for data collection on a Talos Arctica (2-4 days for each transcription complex) or a Titan Krios (1-2 days for each transcription complex) for high-resolution structure determination.

Project Details

Start Date
2019-04-15
End Date
2020-01-31
Status
Closed

Team

Principal Investigator

Richard Ebright
Institution
Rutgers University

Team Members

Vadim Molodtsov
Institution
Rutgers University

Drew Gingerich
Institution
Oregon Health & Science University

Chengyuan Wang
Institution
Rutgers University

Related Publications

Structural basis of transcription-translation coupling Chengyuan Wang, Vadim Molodtsov, Emre Firlar, Jason Kaelber, Gregor Blaha, Min Su, Richard H. Ebright doi: https://doi.org/10.1101/2020.03.01.972380 https://www.biorxiv.org/content/10.1101/2020.03.01.972380v1.full.pdf