J Mol Biol 1996 Mar 01;2563:458-72. doi: 10.1006/jmbi.1996.0101.

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

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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 oxidized nucleosomal templates are found to be identical to within +/- 10%. We conclude that nucleosomes having covalently bound H3 molecules are substrates for transcription, suggesting that the splitting of the nucleosome postulated in the lexosome model of nucleosome transcription is not a necessary event. Interesting future applications of this methodology are discussed.

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Genes referenced: polr3a