Mechanisms of transcription fidelity in prokaryotes and eukaryotes

控制转录保真度的机制是通过基于结构数据的位点定向突变来解决的。最近有报道称，酵母Pol II的Rpb1亚基His1085的取代，是与传入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的结合，但对不匹配和2-dNTPs的结合影响较小。Holmes等人瞄准了EcRNAP β亚基中的Arg678和Asp814残基，这些残基被预测对ntp相关金属离子在活性位点的协调至关重要。然而，这些残基的突变不影响转录。显然，Pol II/EcRNAP结构的复杂性似乎限制了结构驱动的位点定向诱变识别功能上有意义突变的能力。因此，替代结构驱动的位点定向诱变可能是有益的。

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.