Abstract
In bacteria, RNA polymerase (RNAP) often pauses during the early stages of transcription initiation. The structural basis for these transient pauses remains unclear. Here, we present cryo-electron microscopy (cryo-EM) structures of the paused initiation complex (PIC) and initiation complex (IC) of Mycobacterium tuberculosis (Mtb), which include the RNAP core enzyme, the ECF σ factor σE, transcription factor CarD, promoter DNA, and nascent RNA. Our structures with pre-melted scaffolds reveal an intermediate at the 6–7 nt stage compatible with a paused-like intermediate, associated with steric hindrance between the emerging RNA and the σ3.2 region. This clash triggers a swivel of the RNAP structural module and scrunching of the transcription bubble. We also observe positional rearrangement of the σ4 domain, suggesting a poised pre-escape state. In addition, complementary reconstructions with fully matched DNA scaffolds (N-IC and N-PIC) support the physiological relevance of the captured intermediates. Together, our results support the existence of a mechanistic checkpoint during transcription initiation and suggest an RNA-induced model how RNAP conformational dynamics regulate early transcription.
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Data availability
The Cryo-EM density maps and structures have been deposited into the Protein Data Bank (PDB) and Electron Microscopy Data Bank (EMDB) with the accession numbers 9M98 and EMD-63729 for Mtb IC6, 9M9D and EMD-63730 for Mtb IC7, 9M9E and EMD-63731 for Mtb PIC7, EMD-66362 for N-PIC7, EMD-66363 for N-IC6, and EMD-66364 for N-IC7. The PDB entries 8E8M, 8E82, 5UH5, 5ZX2, 6DVD, 6EDT, 6JBQ and 6JCY have been used for structure comparison in this study. Source data are provided with this paper.
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Acknowledgements
We thank the staff members at the National Facility for Protein Science in Shanghai Zhangjiang Lab and the cryo-EM centers of CAS Center for Excellence in Molecular Plant Sciences for their technical assistance on cryo-EM data collection. This study was funded by grants from the National Natural Science Foundation of China (32500154 to L.Z.; 32301018 and 32571394 to K.X.), Shanghai Sailing Program (23YF1450100 to L.Z.), Shanghai Health Commission Clinical Research Special Youth Project (20234Y0116 to L.Z.), Tongji University Independent Original Basic Research Project (22120240275 to L.Z.), the National Key Research and Development Program of China (2021YFA1302200 to K.X.), and the Thousand Talents Plan-Youth to K.X., Fundamental Research Funds for the Central Universities (Tongji University) to K.X.
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L.Z. conceived of the project and designed the experiments. L.Z. performed the sample purification and complex assembly. L.Z. collected the cryo-EM data. L.Z. performed Fluorescence-detecting in vitro transcription assays. L.Z. and K.X. analyzed the data. L.Z. and K.X. wrote the manuscript. All authors discussed the experiments, read and approved the manuscript.
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Zheng, L., Xu, K. Structural basis of pausing during transcription initiation in mycobacterium tuberculosis. Nat Commun (2026). https://doi.org/10.1038/s41467-026-69104-w
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DOI: https://doi.org/10.1038/s41467-026-69104-w
