Protacio R U, Widom J
Department of Biochemistry, Northwestern University, Evanston, IL 60208-3500, USA.
J Mol Biol. 1996 Mar 1;256(3):458-72. doi: 10.1006/jmbi.1996.0101.
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.
我们报告了一种用于体外核小体转录研究的替代方法的开发。该模型系统使我们能够实时、同步且单次跟踪RNA聚合酶分子在通过包装在核小体中的DNA时的进程。使用噬菌体T7 RNA聚合酶和由天然组蛋白或其中两个H3 Cys 110巯基已被氧化形成二硫键的组蛋白制备的重组核小体获得结果。作为时间函数产生的放射性标记转录本的长度和浓度提供了对裸DNA和核小体颗粒上转录速度的直接测量，并允许确定起始、延伸和完成的相对和绝对效率。这些直接测量揭示了延伸过程的新特征。核小体模板上的延伸速度比裸DNA上的速度略慢但可重复。发现这种差异是由于核小体模板上的暂停略有增加。值得注意的是，核小体模板上这种增加的暂停位点也是裸DNA上的暂停位点。发现天然或氧化核小体模板上的延伸速度在±10%范围内相同。我们得出结论，具有共价结合H3分子的核小体是转录的底物，这表明在核小体转录的lexosome模型中假设的核小体分裂不是一个必要事件。讨论了该方法有趣的未来应用。
