Mechanisms of transcription fidelity in prokaryotes and eukaryotes

通过基于结构数据的定点诱变来解决控制转录保真度的机制。最近有报道称，酵母 Pol II 的 Rpb1 亚基中的 His1085 取代是参与与传入 NTP 的 β 和 γ 磷酸盐直接接触的移动元件的一部分，强烈抑制匹配 NTP 的掺入，但对不匹配和 2-dNTP 的掺入影响较小。福尔摩斯等人。靶向 EcRNAP β 亚基中的 Arg678 和 Asp814 残基，预计这些残基对于活性位点中 NTP 相关金属离子的协调至关重要。然而，这些残基的突变并不影响转录。显然，Pol II/EcRNAP 结构的复杂性似乎限制了结构驱动的定点诱变鉴定具有功能意义的突变的能力。因此，结构驱动的定点诱变的替代方案可能会提供信息。

Mechanisms controlling transcription fidelity were addressed by site directed mutagenesis based on the structural data. It has been recently reported that substitutions of His1085 in the Rpb1 subunit of yeast Pol II, which is a part of a mobile element involved in a direct contact with beta and gamma phosphates of the incoming NTP, strongly inhibit incorporation of the matched NTP, but have lesser effect on incorporation of the mismatched and 2-dNTPs. Holmes et al. targeted the Arg678 and Asp814 residues in the beta subunit of EcRNAP that were predicted to be crucial for the coordination of the NTP-associated metal ion in the active site. However, mutation of these residues did not affect transcription. Evidently, the complexity of the Pol II/EcRNAP structure appears to limit the capacity of the structure-driven site-directed mutagenesis for identification of functionally meaningful mutations. Therefore, alternatives to the structure-driven site-directed mutagenesis might be informative.