Szentirmay M N, Sawadogo M
Department of Molecular Genetics, University of Texas, M.D. Anderson Cancer Center, Houston 77030.
Nucleic Acids Res. 1994 Dec 11;22(24):5341-6. doi: 10.1093/nar/22.24.5341.
There are indications that different concentrations of Sarkosyl can block transcription initiation by RNA polymerase II in vitro at different functional steps [Hawley and Roeder (1985) J. Biol. Chem. 260, 8163-8172]. Consequently, this reagent could be a very useful tool for mechanistic studies. So far, however, evidence for the selectivity of Sarkosyl effects on RNA polymerase II transcription has been only indirect. To directly investigate the effect of Sarkosyl on transcription initiation and reinitiation by RNA polymerase II, we employed the reinitiation assay based on utilization of templates containing G-free cassettes (colliding polymerases reinitiation assay, or CoPRA). These experiments showed unambiguously that, under the appropriate conditions, Sarkosyl can be used to block transcription reinitiation by RNA polymerase II while allowing a first round of initiations from preassembled initiation complexes. This inhibition is not due to a disruption of the SII-dependent elongation of the reinitiated transcripts, and the levels of Sarkosyl that prevent transcription reinitiation coincide with the levels that block preinitiation complex assembly. However, Sarkosyl addition to transcription reactions reconstituted with partially purified transcription factors was found to have several undesirable side effects. The usefulness and limitations of the Sarkosyl-based and CoPRA assays for measurements of transcription reinitiation are discussed.
有迹象表明，不同浓度的十二烷基肌氨酸钠能够在体外不同功能步骤阻断RNA聚合酶II的转录起始过程[霍利和罗德（1985年）《生物化学杂志》260, 8163 - 8172]。因此，该试剂可能是进行机制研究的一个非常有用的工具。然而，到目前为止，关于十二烷基肌氨酸钠对RNA聚合酶II转录影响的选择性证据仅为间接证据。为了直接研究十二烷基肌氨酸钠对RNA聚合酶II转录起始和重新起始的影响，我们采用了基于使用含无G盒模板的重新起始分析方法（碰撞聚合酶重新起始分析，或CoPRA）。这些实验明确表明，在适当条件下，十二烷基肌氨酸钠可用于阻断RNA聚合酶II的转录重新起始，同时允许从预组装的起始复合物进行第一轮起始。这种抑制并非由于重新起始转录本的依赖于SII的延伸被破坏，并且阻止转录重新起始的十二烷基肌氨酸钠水平与阻断起始前复合物组装的水平一致。然而，发现在用部分纯化的转录因子重构的转录反应中添加十二烷基肌氨酸钠会产生一些不良副作用。讨论了基于十二烷基肌氨酸钠和CoPRA分析方法在测量转录重新起始方面的实用性和局限性。
