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.

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Imashimizu, Masahiko; Takahashi, Hiroki; Oshima, Taku et al. (2015) Visualizing translocation dynamics and nascent transcript errors in paused RNA polymerases in vivo. Genome Biol 16:98
Penno, Christophe; Sharma, Virag; Coakley, Arthur et al. (2015) Productive mRNA stem loop-mediated transcriptional slippage: Crucial features in common with intrinsic terminators. Proc Natl Acad Sci U S A 112:E1984-93
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Imashimizu, Masahiko; Kashlev, Mikhail (2014) Unveiling translocation intermediates of RNA polymerase. Proc Natl Acad Sci U S A 111:7507-8
Ishibashi, Toyotaka; Dangkulwanich, Manchuta; Coello, Yves et al. (2014) Transcription factors IIS and IIF enhance transcription efficiency by differentially modifying RNA polymerase pausing dynamics. Proc Natl Acad Sci U S A 111:3419-24
Imashimizu, Masahiko; Shimamoto, Nobuo; Oshima, Taku et al. (2014) Transcription elongation. Heterogeneous tracking of RNA polymerase and its biological implications. Transcription 5:e28285

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