RNA. 2014 May;20(5):644-55. doi: 10.1261/rna.040444.113. Epub 2014 Mar 10.
Many RNAs are known to act as regulators of transcription in eukaryotes, including certain small RNAs that directly inhibit RNA polymerases both in prokaryotes and eukaryotes. We have examined the potential for a variety of RNAs to directly inhibit transcription by yeast RNA polymerase II (Pol II) and find that unstructured RNAs are potent inhibitors of purified yeast Pol II. Inhibition by RNA is achieved by blocking binding of the DNA template and requires binding of the RNA to Pol II prior to open complex formation. RNA is not able to displace a DNA template that is already stably bound to Pol II, nor can RNA inhibit elongating Pol II. Unstructured RNAs are more potent inhibitors than highly structured RNAs and can also block specific transcription initiation in the presence of basal transcription factors. Crosslinking studies with ultraviolet light show that unstructured RNA is most closely associated with the two large subunits of Pol II that comprise the template binding cleft, but the RNA has contacts in a basic residue channel behind the back wall of the active site. These results are distinct from previous observations of specific inhibition by small, structured RNAs in that they demonstrate a sensitivity of the holoenzyme to inhibition by unstructured RNA products that bind to a surface outside the DNA cleft. These results are discussed in terms of the need to prevent inhibition by RNAs, either though sequestration of nascent RNA or preemptive interaction of Pol II with the DNA template.
许多 RNA 被认为在真核生物中转录调控因子，包括某些小 RNA 直接抑制原核生物和真核生物中的 RNA 聚合酶。我们已经研究了各种 RNA 直接抑制酵母 RNA 聚合酶 II (Pol II) 转录的潜力，发现无结构 RNA 是纯化酵母 Pol II 的有效抑制剂。RNA 通过阻断 DNA 模板的结合来抑制转录，并且需要在开放复合物形成之前将 RNA 结合到 Pol II。RNA 不能置换已经稳定结合到 Pol II 的 DNA 模板，也不能抑制延伸的 Pol II。无结构 RNA 比高度结构化 RNA 更有效抑制剂，并且在存在基础转录因子的情况下也可以阻断特定的转录起始。用紫外线进行的交联研究表明，无结构 RNA 与构成模板结合裂隙的 Pol II 的两个大亚基最密切相关，但 RNA 在活性位点后壁后面的碱性残基通道中具有接触。这些结果与先前观察到的小、结构化 RNA 特异性抑制不同，因为它们表明全酶对结合到 DNA 裂隙外表面的无结构 RNA 产物的抑制敏感。这些结果根据需要讨论了通过隔离新生 RNA 或 Pol II 与 DNA 模板的抢先相互作用来防止 RNA 抑制的问题。
