Edited by Jeffrey Roberts, Cornell University, Ithaca, NY; received September 19, 2022; accepted January 10, 2023
February 16, 2023
120 (8) e2215945120
Significance
Transcriptional pausing provides a hub for gene regulation. Pausing provides a timing mechanism to coordinate regulatory interactions, cotranscriptional RNA folding and protein synthesis, and stop signals for transcriptional termination. Cellular RNA polymerases (RNAPs) are complex, with multiple mobile modules shifting positions to control its catalytic activity and pause RNAP in response to DNA-encoded pause signals. Understanding how these modules move to enable pausing is crucial for a mechanistic understanding of gene regulation. Our results clarify the picture significantly by defining multiple states among which paused RNAP partitions in response to different pause signals. This work contributes to an emerging theme wherein multiple interconverting states of the RNAP proceed through a pathway (e.g., initiation or pausing), providing multiple opportunities for regulation.
Abstract
Transcriptional pausing underpins the regulation of cellular RNA synthesis, but its mechanism remains incompletely understood. Sequence-specific interactions of DNA and RNA with the dynamic, multidomain RNA polymerase (RNAP) trigger reversible conformational changes at pause sites that temporarily interrupt the nucleotide addition cycle. These interactions initially rearrange the elongation complex (EC) into an elemental paused EC (ePEC). ePECs can form longer-lived PECs by further rearrangements or interactions of diffusible regulators. For both bacterial and mammalian RNAPs, a half-translocated state in which the next DNA template base fails to load into the active site appears central to the ePEC. Some RNAPs also swivel interconnected modules that may stabilize the ePEC. However, it is unclear whether swiveling and half-translocation are requisite features of a single ePEC state or if multiple ePEC states exist. Here, we use cryo-electron microscopy (cryo-EM) analysis of ePECs with different RNA–DNA sequences combined with biochemical probes of ePEC structure to define an interconverting ensemble of ePEC states. ePECs occupy either pre- or half-translocated states but do not always swivel, indicating that difficulty in forming the posttranslocated state at certain RNA–DNA sequences may be the essence of the ePEC. The existence of multiple ePEC conformations has broad implications for transcriptional regulation.
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Data, Materials, and Software Availability
Cryo-EM data have been deposited in the RCSB Protein Data Bank (www.pdb.org) and in the Electron Microscopy Data Bank (www.emdatabank.org). The PDB accession codes for the coordinates of con-ePEC−1, con-ePEC_fTL, con-ePEC_ufTL, his-ePEC_fTL-Fin1, his-ePEC-fTL-Fin2, his-ePEC_fTL-Fout, his-ePEC_ufTL1, and his-ePEC_ufTL2 are 8EG7 (63), 8EG8 (64), 8EGB (65), 8EH8 (66), 8EH9 (67), 8EHA (68), 8EHF (69), and 8EHI (70), respectively, and the accession codes for the cryo-EM maps are EMD-28109 (71), EMD-28110 (72), EMD-28113 (73), EMD-28143 (74), EMD-28144 (75), EMD-28145 (76), EMD-28146 (77), and EMD-28148 (78), respectively. All study data are included in the article, SI Appendix, or both. Previously published data were used for this work (DOI: https://doi.org/10.1073/pnas.2101805118).
Acknowledgments
We thank members of the Darst-Campbell and Landick Laboratories for experimental advice and helpful discussions and M. Ebrahim, J. Sotiris, and H. Ng at The Rockefeller University Evelyn Gruss Lipper Cryo-electron Microscopy Resource Center for help with cryo-EM data collection. This work was supported by Burroughs Wellcome Fund CASI award 1016945 to T.V.M. and NIH grants R35 GM118130 to S.A.D and R01 GM38330 to R.L.
Author contributions
J.Y.K., T.V.M., Y.B., J.C., S.A.D., and R.L. designed research; J.Y.K., T.V.M., Y.B., E.L., J.L., and R.L. performed research; J.Y.K., T.V.M., Y.B., J.C., J.L., S.A.D., and R.L. analyzed data; and J.Y.K., T.V.M., Y.B., J.C., S.A.D., and R.L. wrote the paper.
Competing interests
The authors declare no competing interest.
Supporting Information
Movie S1.
The movie shows the movements of active-site modules (BH, TL, RH, FL) and nucleic acids that connect the different elemental PEC states observed for con-ePEC and his-ePEC in two side-by-side views of the active site of RNAP. Movements of the BH, RH, and FL that accompany TL folding and unfolding are also likely to occur during the active NAC.
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Information & Authors
Information
Published in
Proceedings of the National Academy of Sciences
Vol. 120 | No. 8
February 21, 2023
Classifications
Copyright
Data, Materials, and Software Availability
Cryo-EM data have been deposited in the RCSB Protein Data Bank (www.pdb.org) and in the Electron Microscopy Data Bank (www.emdatabank.org). The PDB accession codes for the coordinates of con-ePEC−1, con-ePEC_fTL, con-ePEC_ufTL, his-ePEC_fTL-Fin1, his-ePEC-fTL-Fin2, his-ePEC_fTL-Fout, his-ePEC_ufTL1, and his-ePEC_ufTL2 are 8EG7 (63), 8EG8 (64), 8EGB (65), 8EH8 (66), 8EH9 (67), 8EHA (68), 8EHF (69), and 8EHI (70), respectively, and the accession codes for the cryo-EM maps are EMD-28109 (71), EMD-28110 (72), EMD-28113 (73), EMD-28143 (74), EMD-28144 (75), EMD-28145 (76), EMD-28146 (77), and EMD-28148 (78), respectively. All study data are included in the article, SI Appendix, or both. Previously published data were used for this work (DOI: https://doi.org/10.1073/pnas.2101805118).
Submission history
Received: September 19, 2022
Accepted: January 10, 2023
Published online: February 16, 2023
Published in issue: February 21, 2023
Keywords
- RNA polymerase
- transcriptional pausing
- transcriptional regulation
- cryo-EM
- Escherichia coli
Acknowledgments
We thank members of the Darst-Campbell and Landick Laboratories for experimental advice and helpful discussions and M. Ebrahim, J. Sotiris, and H. Ng at The Rockefeller University Evelyn Gruss Lipper Cryo-electron Microscopy Resource Center for help with cryo-EM data collection. This work was supported by Burroughs Wellcome Fund CASI award 1016945 to T.V.M. and NIH grants R35 GM118130 to S.A.D and R01 GM38330 to R.L.
Author Contributions
J.Y.K., T.V.M., Y.B., J.C., S.A.D., and R.L. designed research; J.Y.K., T.V.M., Y.B., E.L., J.L., and R.L. performed research; J.Y.K., T.V.M., Y.B., J.C., J.L., S.A.D., and R.L. analyzed data; and J.Y.K., T.V.M., Y.B., J.C., S.A.D., and R.L. wrote the paper.
Competing Interests
The authors declare no competing interest.
Notes
This article is a PNAS Direct Submission.
Authors
Affiliations
Department of Chemistry, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea
Tatiana V. Mishanina1
Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA 92093
Department of Biochemistry, University of Wisconsin–Madison, Madison, WI 53706
James Chen
Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY 10065
Present address: New York University (NYU) Grossman School of Medicine, New York, NY 10016.
Eliza Llewellyn
Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY 10065
Present address: Beagle Bioscience, Westport, CT 06881.
James Liu
Department of Biochemistry, University of Wisconsin–Madison, Madison, WI 53706
Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY 10065
Department of Biochemistry, University of Wisconsin–Madison, Madison, WI 53706
Department of Bacteriology, University of Wisconsin–Madison, Madison, WI 53706
Notes
1
J.Y.K., T.V.M., and Y.B. contributed equally to this work.
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