Mechanisms of transcription fidelity in prokaryotes and eukaryotes

在结构数据的基础上，通过定点突变解决了控制转录保真度的机制。最近有报道，酵母POL II的Rpb1亚基上的His1085的替换强烈地抑制了匹配的NTP的掺入，但对错配的和2-dNTPs的掺入的影响较小。Holmes等人。靶向EcRNAPβ亚基中的Arg678和Asp814残基，这两个残基被预测对NTP相关金属离子在活性部位的配位至关重要。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突变识别有功能意义的突变的能力。因此，替代结构驱动的定点突变可能是有用的。

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.