NUCLEOSOME TRANSCRIPTION STUDIED IN A REAL-TIME SYNCHRONOUS SYSTEM - TEST OF THE LEXOSOME MODEL AND DIRECT MEASUREMENT OF EFFECTS DUE TO HISTONE OCTAMER

We report the development of an alternative approach to studies on nuc leosome transcription in vitro. This model system allows us to follow the real-time, synchronous and single passage of RNA polymerase molecu les 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 le ngths and concentrations of radiolabeled transcripts produced as a fun ction of time provide direct measurements of the velocities of transcr iption on naked DNA and on the nucleosomal particles, and allow both r elative and absolute efficiencies of initiation, elongation and comple tion to be determined. These direct measurements reveal new features o f the elongation process. The velocities of elongation on the nucleoso mal templates are slightly but reproducibly slower than those on naked DNA. This difference is found to be due to a slight increase in pausi ng on the nucleosomal templates. Remarkably, the sites of this increas ed pausing on the nucleosomal templates are also pause sites on the na ked DNA. The velocities of elongation on native or oxidized nucleosoma l templates are found to be identical to within +/- 10%. We conclude t hat nucleosomes having covalently bound H3 molecules are substrates fo r transcription, suggesting that the splitting of the nucleosome postu lated in the lexosome model of nucleosome transcription is not a neces sary event. Interesting future applications of this methodology are di scussed. (C) 1996 Academic Press Limited