Promoter unwinding and promoter clearance by RNA polymerase: Detection by single-molecule DNA nanomanipulation

Revyakin, A., Ebright, R. H., Strick, T. R. (April 2004) Promoter unwinding and promoter clearance by RNA polymerase: Detection by single-molecule DNA nanomanipulation. Proceedings of the National Academy of Sciences of the United States of America, 101 (14). pp. 4776-4780. ISSN 0027-8424

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URL: https://www.ncbi.nlm.nih.gov/pubmed/15037753
DOI: 10.1073/pnas.0307241101

Abstract

By monitoring the end-to-end extension of a mechanically stretched, supercoiled, single DNA molecule, we have been able directly to observe the change in extension associated with unwinding of approximately one turn of promoter DNA by RNA polymerase (RNAP). By performing parallel experiments with negatively and positively supercoiled DNA, we have been able to deconvolute the change in extension caused by RNAP-dependent DNA unwinding (with approximate to1-bp resolution) and the change in extension caused by RNAP-dependent DNA compaction (with approximate to5-nm resolution). We have used this approach to quantify the extent of unwinding and compaction, the kinetics of unwinding and compaction, and effects of supercoiling, sequence, ppGpp, and nucleotides. We also have used this approach to detect promoter clearance and promoter recycling by successive RNAP molecules. We find that the rate of formation and the stability of the unwound complex depend profoundly on supercoiling and that supercoiling exerts its effects mechanically (through torque), and not structurally (through the number and position of supercoils). The approach should permit analysis of other nucleic-acid-processing factors that cause changes in DNA twist and/or DNA compaction.

Item Type: Paper
Uncontrolled Keywords: ESCHERICHIA-COLI escherichia coli escherichia_coli E-Coli SUPERCOILED DNA supercoiled DNA TRANSCRIPTION INITIATION transcription initiation STRUCTURAL structural ORGANIZATION organization OPEN COMPLEX open complex MECHANISM mechanism
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > enzymes > RNA polymerase
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > DNA expression > promoter
CSHL Authors:
Communities: CSHL labs > Strick lab
Depositing User: CSHL Librarian
Date: April 2004
Date Deposited: 26 Jan 2012 21:28
Last Modified: 11 Jan 2018 21:13
Related URLs:
URI: http://repository.cshl.edu/id/eprint/22467

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