Mechanisms of transcription fidelity in prokaryotes and eukaryotes

原核生物和真核生物转录保真度的机制

• 批准号: 8763224
• 负责人: MIKHAIL KASHLEV
• 金额: $ 54.38万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8763224
• 关键词: Active Sites; Address; Affect; Aging; Biochemical; Biochemistry; Biological; Biology; Cells; DNA; DNA biosynthesis; DNA-Directed RNA Polymerase; Data; Elements; Enzymes; Escherichia coli; Eukaryota; Frequencies; Gene Expression; Genetic Transcription; Genome Stability; Human; In Vitro; Ions; Lead; Libraries; Malignant Neoplasms; Messenger RNA; Metals; Methods; Mutagenesis; Mutation; Nature; Organism; Phenotype; Polymerase; Prokaryotic Cells; Proteins; RNA; RNA Polymerase II; Reporting; Saccharomyces cerevisiae; Site-Directed Mutagenesis; Structure; Techniques; Tertiary Protein Structure; Yeasts; azauracil; base; genetic selection; improved; in vivo; inorganic phosphate; insertion/deletion mutation; mutant; screening; tool; transcription factor S-II; transcriptome sequencing

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.

基于结构数据通过定点诱变解决控制转录保真度的机制。最近有报道称，酵母Pol II的Rpb 1亚基中His 1085的取代强烈抑制了匹配NTP的掺入，但对错配和2-dNTP的掺入影响较小，所述Rpb 1亚基是参与与引入NTP的β和γ磷酸盐直接接触的移动的元件的一部分。Holmes等人靶向了EcRNAP β亚基中的Arg 678和Asp 814残基，这些残基被预测对活性位点中NTP相关金属离子的配位至关重要。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诱变用于鉴定功能上有意义的突变的能力。因此，结构驱动的定点诱变的替代方案可能是有益的。