期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 289, 期 35, 页码 24549-24559出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M114.584037
关键词
Promoter; RNA Polymerase; Transcription; Transcription Initiation Factor; X-ray Crystallography
资金
- National Institutes of Health [GM087350-A1]
- Spanish government [BFU2010-16336]
- Russian Foundation for Basic Research [14-04-01696, 14-04-31994]
- Russian Academy of Sciences Presidium Program in Molecular and Cellular Biology
Background: Cellular RNA polymerases start transcription by de novo RNA priming. Results: Structures and biochemical studies of initially transcribing complexes elucidate the de novo transcription initiation and early stage of RNA transcription. Conclusion: 5-end of RNA in the transcribing complex starts sigma ejection from core enzyme. Significance: Insights from this study can be applicable to all cellular RNA polymerases. The bacterial RNA polymerase (RNAP) holoenzyme containing sigma factor initiates transcription at specific promoter sites by de novo RNA priming, the first step of RNA synthesis where RNAP accepts two initiating ribonucleoside triphosphates (iNTPs) and performs the first phosphodiester bond formation. We present the structure of de novo transcription initiation complex that reveals unique contacts of the iNTPs bound at the transcription start site with the template DNA and also with RNAP and demonstrate the importance of these contacts for transcription initiation. To get further insight into the mechanism of RNA priming, we determined the structure of initially transcribing complex of RNAP holoenzyme with 6-mer RNA, obtained by in crystallo transcription approach. The structure highlights RNAP-RNA contacts that stabilize the short RNA transcript in the active site and demonstrates that the RNA 5-end displaces sigma region 3.2 from its position near the active site, which likely plays a key role in sigma ejection during the initiation-to-elongation transition. Given the structural conservation of the RNAP active site, the mechanism of de novo RNA priming appears to be conserved in all cellular RNAPs.
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