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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010795-07
Application #
8763224
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2013
Total Cost
$543,787
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
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
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
Vitiello, Christal L; Kireeva, Maria L; Lubkowska, Lucyna et al. (2014) Coliphage HK022 Nun protein inhibits RNA polymerase translocation. Proc Natl Acad Sci U S A 111:E2368-75
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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
Afonin, Kirill A; Kasprzak, Wojciech K; Bindewald, Eckart et al. (2014) In silico design and enzymatic synthesis of functional RNA nanoparticles. Acc Chem Res 47:1731-41
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Parks, Adam R; Court, Carolyn; Lubkowska, Lucyna et al. (2014) Bacteriophage λ N protein inhibits transcription slippage by Escherichia coli RNA polymerase. Nucleic Acids Res 42:5823-9
Imashimizu, Masahiko; Shimamoto, Nobuo; Oshima, Taku et al. (2014) Transcription elongation: Heterogeneous tracking of RNA polymerase and its biological implications. Transcription 5:
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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