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dc.contributor.authorFang, Chengli
dc.contributor.authorLi, Lingting
dc.contributor.authorShen, Liqiang
dc.contributor.authorShi, Jing
dc.contributor.authorwang, sheng
dc.contributor.authorFeng, Yu
dc.contributor.authorZhang, Yu
dc.date.accessioned2019-08-01T13:15:42Z
dc.date.available2019-08-01T13:15:42Z
dc.date.issued2019-05-27
dc.identifier.doi10.1093/nar/gkz470
dc.identifier.urihttp://hdl.handle.net/10754/656303
dc.description.abstractBacterial RNA polymerase (RNAP) forms distinct holoenzymes with extra-cytoplasmic function (ECF) σ factors to initiate specific gene expression programs. In this study, we report a cryo-EM structure at 4.0 Å of Escherichia coli transcription initiation complex comprising σE—the most-studied bacterial ECF σ factor (Ec σE-RPo), and a crystal structure at 3.1 Å of Mycobacterium tuberculosis transcription initiation complex with a chimeric σH/E (Mtb σH/E-RPo). The structure of Ec σE-RPo reveals key interactions essential for assembly of E. coli σE-RNAP holoenzyme and for promoter recognition and unwinding by E. coli σE. Moreover, both structures show that the non-conserved linkers (σ2/σ4 linker) of the two ECF σ factors are inserted into the active-center cleft and exit through the RNA-exit channel. We performed secondary-structure prediction of 27,670 ECF σ factors and find that their non-conserved linkers probably reach into and exit from RNAP active-center cleft in a similar manner. Further biochemical results suggest that such σ2/σ4 linker plays an important role in RPo formation, abortive production and promoter escape during ECF σ factors-mediated transcription initiation.
dc.description.sponsorshipWe thank Prof. Richard Ebright for generous gifts of pACYC-duet-Mtb-rpoA-rpoD, pETDuet-Mtb-rpoB-rpoC, Prof. Bryce Nickels for generous gift of pET28c-Ecσ70, Prof. Xiaoming Zhang for generous gift of M. tuberculosis genomic DNA and Tolo Biotechnology for generous gift of pTolo-EX vectors. We thank the staff at beamline BL18U1/BL19U1 of National Center for Protein Science Shanghai (NCPSS), and at beamline BL17U1 of Shanghai Synchrotron Radiation Facility for assistance during data collection. We thank Shenghai Chang at center of cryo Electron Microscopy for help with cryo-EM sample preparation and data collection. We thank the state key laboratory of bio-organic and natural products chemistry at Shanghai institute of organic chemistry at CAS for sharing the stopped-flow fluorescence spectrometer
dc.description.sponsorshipFUNDING Strategic Priority Research Program of the Chinese Academy of Sciences [XDB29020000]; National Natural Science Foundation of China [31670067, 31822001]; Leading Science Key Research Program of the Chinese Academy of Sciences [QYZDB-SSW-SMC005]. Funding for open access charge: Chinese Academy of Sciences (QYZDB-SSW-SMC005). Conflict of interest statement. None declared
dc.publisherOxford University Press (OUP)
dc.rightsThis is a pre-copyedited, author-produced PDF of an article accepted for publication in Nucleic Acids Research following peer review. The version of record is available online at: .
dc.titleStructures and mechanism of transcription initiation by bacterial ECF factors
dc.typeArticle
dc.contributor.departmentComputer, Electrical and Mathematical Sciences and Engineering (CEMSE) Division
dc.identifier.journalNucleic Acids Research
dc.eprint.versionPublisher's Version/PDF
dc.contributor.institutionKey Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China
dc.contributor.institutionUniversity of Chinese Academy of Sciences, Beijing 100049, China
dc.contributor.institutionDepartment of Biochemistry and Molecular Biology, School of Medicine, Zhejiang University, Hangzhou 310058, China
kaust.personWang, Sheng
refterms.dateFOA2019-08-01T13:17:36Z


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