Structures and mechanism of transcription initiation by bacterial ECF factors
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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.
SponsorsWe 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
FUNDING 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
PublisherOxford University Press (OUP)
JournalNucleic Acids Research