Equilibrium and stopped-flow kinetic studies of interaction between T7 RNA polymerase and its promoters measured by protein and 2-aminopurine fluorescence changes.

The Journal of Biological Chemistry
Y JiaSmita S Patel

Abstract

The mechanism of bacteriophage T7 RNA polymerase binding to its promoter DNA was investigated using stopped-flow and equilibrium methods. To measure the kinetics of protein-DNA interactions in real time, changes in tryptophan fluorescence in the polymerase and 2-aminopurine (2-AP) fluorescence in the promoter DNA upon binary complex formation were used as probes. The protein fluorescence changes measured conformational changes in the polymerase whereas the fluorescence changes of 2-AP base, substituted in place of dA in the initiation region (-4 to +4), measured structural changes in the promoter DNA, such as DNA melting. The kinetic studies, carried out in the absence of the initiating nucleotide, are consistent with a two-step DNA binding mechanism, [formula: see text] where the RNA polymerase forms an initial weak EDa complex rapidly with an equilibrium association constant K1. The EDa complex then undergoes a conformational change to EDb, wherein RNA polymerase is specifically and tightly bound to the promoter DNA. Both the polymerase and the promoter DNA may undergo structural changes during this isomerization step. The isomerization of EDa to EDb is a fast step relative to the rate of transcription initiation and its rate...Continue Reading

References

Feb 6, 1979·Biochemistry·J L OakleyJ E Coleman
Feb 14, 1992·Science·M Kainz, J Roberts
Jan 1, 1987·CRC Critical Reviews in Biochemistry·A A Travers
Aug 18, 1981·Biochemistry·H L Osterman, J E Coleman
Apr 1, 1984·Proceedings of the National Academy of Sciences of the United States of America·P DavanlooF W Studier
Feb 25, 1984·Journal of Molecular Biology·B A MoffattF W Studier
Mar 5, 1983·Journal of Molecular Biology·S E PhillipsH Wajcman
Jun 5, 1983·Journal of Molecular Biology·J J Dunn, F W Studier
Apr 26, 1996·Journal of Molecular Biology·X ZhangR Schleif
Jun 1, 1996·Current Opinion in Structural Biology·D P Millar
Nov 30, 1967·Biochemical and Biophysical Research Communications·H Hayatsu, T Ukita

❮ Previous
Next ❯

Citations

Feb 3, 2011·Journal of Neuroimmune Pharmacology : the Official Journal of the Society on NeuroImmune Pharmacology·Kathryn E Medders, Marcus Kaul
Nov 8, 2002·Journal of Molecular Biology·Rajiv P Bandwar, Smita S Patel
Feb 13, 2002·Biosensors & Bioelectronics·Christine N Jayarajah, Michael Thompson
Sep 13, 2001·Bioorganic & Medicinal Chemistry·J A Beckingham, G D Glick
Feb 16, 2013·ACS Synthetic Biology·Katherine A RiccioneLingchong You
Dec 19, 2009·Proceedings of the National Academy of Sciences of the United States of America·Guo-Qing TangSmita S Patel
Oct 15, 1998·Proceedings of the National Academy of Sciences of the United States of America·L ShapiroC A Dinarello
Mar 31, 2010·The Journal of Biological Chemistry·Maria F LodeiroCraig E Cameron
Jan 1, 2009·The Journal of Biological Chemistry·Guo-Qing TangSmita S Patel
Mar 21, 1998·Nucleic Acids Research·B HolzE Weinhold
Feb 16, 2013·Science·Sangjin KimX Sunney Xie
Dec 21, 2010·Biochemistry. Biokhimii︠a︡·O S FedorovaD G Knorre
Sep 13, 2012·BMC Bioinformatics·Piotr SetnyMartin Zacharias
Aug 3, 2000·Bioscience, Biotechnology, and Biochemistry·Y C Chen, S T Jeng
Jul 8, 2000·Proceedings of the National Academy of Sciences of the United States of America·R Baliga, D M Crothers
Jan 22, 1998·Proceedings of the National Academy of Sciences of the United States of America·M RongR K Durbin
Oct 6, 2009·Nature Chemical Biology·Cheemeng TanLingchong You
Feb 16, 2011·Biophysical Journal·Gary M SkinnerKoen Visscher
Dec 6, 2005·Biophysical Chemistry·R Murugan
Feb 12, 1998·Journal of Molecular Biology·A Ujvári, C T Martin
Aug 2, 2002·The Journal of Biological Chemistry·Natalie M StanoSmita S Patel
Feb 4, 2011·Chemical Society Reviews·Nan Dai, Eric T Kool
Nov 21, 2002·The Journal of Biological Chemistry·Alexander KukarinWilliam T McAllister

❮ Previous
Next ❯

Related Concepts

Related Feeds

ASBMB Publications

The American Society for Biochemistry and Molecular Biology (ASBMB) includes the Journal of Biological Chemistry, Molecular & Cellular Proteomics, and the Journal of Lipid Research. Discover the latest research from ASBMB here.