Nucleosome transcription studied in a real-time synchronous system: test of the lexosome model and direct measurement of effects due to histone octamer

Journal of Molecular Biology
R U Protacio, Jonathan Widom

Abstract

We report the development of an alternative approach to studies on nucleosome transcription in vitro. This model system allows us to follow the real-time, synchronous and single passage of RNA polymerase molecules as they progress through DNA packaged in nucleosomes. Results are obtained using phage T7 RNA polymerase with reconstituted nucleosomes prepared from native histones or from histones in which the two H3 Cys 110 thiol groups have been oxidized to form a disulfide bridge. The lengths and concentrations of radiolabeled transcripts produced as a function of time provide direct measurements of the velocities of transcription on naked DNA and on the nucleosomal particles, and allow both relative and absolute efficiencies of initiation, elongation and completion to be determined. These direct measurements of reveal new features of the elongation process. The velocities of elongation on the nucleosomal templates are slightly but reproducibly slower than those on naked DNA. This difference is found to be due to a slight increase in pausing on the nucleosomal templates. Remarkably, the sites of this increased pausing on the nucleosomal templates are also pause sites on the naked DNA. The velocities of elongation on native or ox...Continue Reading

Citations

Aug 25, 2005·Cold Spring Harbor Symposia on Quantitative Biology·S ChakravarthyK Luger
Nov 14, 2000·Molecular and Cellular Biology·R U ProtacioJ Widom
Jan 1, 1997·Annual Review of Biochemistry·S M UptainM J Chamberlin
Jul 1, 1998·Annual Review of Biophysics and Biomolecular Structure·J Widom
Feb 18, 1997·Proceedings of the National Academy of Sciences of the United States of America·P T Lowary, J Widom
Feb 22, 2011·Annual Review of Biophysics·Andrew J Andrews, Karolin Luger
May 9, 2012·Genetics Research International·Benjamin AlbertOlivier Gadal
Jul 7, 2007·Molecular Cell·Aurélien BancaudAriel Prunell
Sep 12, 1997·The Journal of Biological Chemistry·C H Chang, D S Luse
Jul 27, 2018·Histochemistry and Cell Biology·Christian Schöfer, Klara Weipoltshammer
Sep 11, 2004·Assay and Drug Development Technologies·Gary M Skinner, Koen Visscher
Jul 30, 2002·Journal of Molecular Biology·Joel M GottesfeldKarolin Luger
Aug 23, 2003·Biochimica Et Biophysica Acta·Miguel A SánchezEnrique Palacián
Oct 31, 2006·Biochimie·Christophe Lavelle
Feb 17, 2009·Structure·Jordanka ZlatanovaKen van Holde
Jan 7, 1998·Science·J Widom

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