TY - JOUR
T1 - Structural basis of template strand deoxyuridine promoter recognition by a viral RNA polymerase
AU - Fraser, Alec
AU - Sokolova, Maria L.
AU - Drobysheva, Arina V.
AU - Gordeeva, Julia V.
AU - Borukhov, Sergei
AU - Jumper, John
AU - Severinov, Konstantin V.
AU - Leiman, Petr G.
N1 - Funding Information:
This work was supported by the Skoltech NGP Program (Skoltech-MIT joint project), the Russian Science Foundation (Grant 19-74-00011 to M.L.S.), the Russian Foundation for Basic Research (Grant 20-34-90079 to J.V.G.), the National Institutes of Health (Grant R01GM130942/Subaward 0518GWB837 to S.B.), the Busch Biomedical Grant Program (K.V.S.), and the Ministry of Science and Higher Education of Russian Federation (Agreement No. 075-10-2021-114 to K.V.S.). The work was also supported by the UTMB Department of Biochemistry and Molecular Biology (A.F., M.L.S., and P.G.L.) and by the UTMB Sealy Center for Structural Biology and Molecular Biophysics (P.G.L.). The MD work was performed using the computing facilities of the Texas Advanced Computing Center (TACC, http://www.tacc.utexas.edu) at The University of Texas for which we are very grateful. We thank the Stanford-SLAC Cryo-EM Facilities, supported by Stanford University, SLAC, and the National Institutes of Health S10 Instrumentation Programs that were used to collect the AR9 nvRNAP holoenzyme cryo-EM data. We acknowledge the use of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. We thank the staff of the LS-CAT Sector 21 beamlines that are supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (Grant 085P1000817). We acknowledge the use of the Berkeley Center for Structural Biology (supported in part by the Howard Hughes Medical Institute) at the Advanced Light Source (a Department of Energy Office of Science User Facility under Contract No. DE-AC02-05CH11231) and we thank the staff of the beamline 5.0.2. Finally, we thank Dr. Mark A. White for his help and assistance with the initial crystallization and X-ray data collection of the AR9 nvRNAP core, Dr. Michael B. Sherman for his help with the cryo-EM data collection, and Dr. Tatyana O. Artamonova for mass-spectrometry analysis of gp226 digestion products. The research reported in this paper extensively used the facilities and resources of the UTMB SCSB Macromolecular Structure X-ray Laboratory and UTMB SCSB Cryo-EM Laboratory. We are grateful to Andriy Kryshtafovych, one of the Critical Assessment of Protein Structure Prediction 14 (CASP 14) competition organizers, and the AlphaFold Team for sharing their predicted models of all five subunits of the AR9 nvRNAP holoenzyme prior to the conclusion of the CASP 14 competition. These models were used in model building and validation of chain tracing of the AR9 nvRNAP holoenzyme. In addition, the atomic model of phage phiKZ gp68 was created with the help of AlphaFold Colab. The full list of the AlphaFold Team members is given in the Supplementary Information with John Jumper representing the entire AlphaFold Team in the author list.
Funding Information:
This work was supported by the Skoltech NGP Program (Skoltech-MIT joint project), the Russian Science Foundation (Grant 19-74-00011 to M.L.S.), the Russian Foundation for Basic Research (Grant 20-34-90079 to J.V.G.), the National Institutes of Health (Grant R01GM130942/Subaward 0518GWB837 to S.B.), the Busch Biomedical Grant Program (K.V.S.), and the Ministry of Science and Higher Education of Russian Federation (Agreement No. 075-10-2021-114 to K.V.S.). The work was also supported by the UTMB Department of Biochemistry and Molecular Biology (A.F., M.L.S., and P.G.L.) and by the UTMB Sealy Center for Structural Biology and Molecular Biophysics (P.G.L.). The MD work was performed using the computing facilities of the Texas Advanced Computing Center (TACC, http://www.tacc.utexas.edu ) at The University of Texas for which we are very grateful. We thank the Stanford-SLAC Cryo-EM Facilities, supported by Stanford University, SLAC, and the National Institutes of Health S10 Instrumentation Programs that were used to collect the AR9 nvRNAP holoenzyme cryo-EM data. We acknowledge the use of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. We thank the staff of the LS-CAT Sector 21 beamlines that are supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (Grant 085P1000817). We acknowledge the use of the Berkeley Center for Structural Biology (supported in part by the Howard Hughes Medical Institute) at the Advanced Light Source (a Department of Energy Office of Science User Facility under Contract No. DE-AC02-05CH11231) and we thank the staff of the beamline 5.0.2. Finally, we thank Dr. Mark A. White for his help and assistance with the initial crystallization and X-ray data collection of the AR9 nvRNAP core, Dr. Michael B. Sherman for his help with the cryo-EM data collection, and Dr. Tatyana O. Artamonova for mass-spectrometry analysis of gp226 digestion products. The research reported in this paper extensively used the facilities and resources of the UTMB SCSB Macromolecular Structure X-ray Laboratory and UTMB SCSB Cryo-EM Laboratory. We are grateful to Andriy Kryshtafovych, one of the Critical Assessment of Protein Structure Prediction 14 (CASP 14) competition organizers, and the AlphaFold Team for sharing their predicted models of all five subunits of the AR9 nvRNAP holoenzyme prior to the conclusion of the CASP 14 competition. These models were used in model building and validation of chain tracing of the AR9 nvRNAP holoenzyme. In addition, the atomic model of phage phiKZ gp68 was created with the help of AlphaFold Colab. The full list of the AlphaFold Team members is given in the with John Jumper representing the entire AlphaFold Team in the author list.
Publisher Copyright:
© 2022, The Author(s).
PY - 2022/12
Y1 - 2022/12
N2 - Recognition of promoters in bacterial RNA polymerases (RNAPs) is controlled by sigma subunits. The key sequence motif recognized by the sigma, the −10 promoter element, is located in the non-template strand of the double-stranded DNA molecule ~10 nucleotides upstream of the transcription start site. Here, we explain the mechanism by which the phage AR9 non-virion RNAP (nvRNAP), a bacterial RNAP homolog, recognizes the −10 element of its deoxyuridine-containing promoter in the template strand. The AR9 sigma-like subunit, the nvRNAP enzyme core, and the template strand together form two nucleotide base-accepting pockets whose shapes dictate the requirement for the conserved deoxyuridines. A single amino acid substitution in the AR9 sigma-like subunit allows one of these pockets to accept a thymine thus expanding the promoter consensus. Our work demonstrates the extent to which viruses can evolve host-derived multisubunit enzymes to make transcription of their own genes independent of the host.
AB - Recognition of promoters in bacterial RNA polymerases (RNAPs) is controlled by sigma subunits. The key sequence motif recognized by the sigma, the −10 promoter element, is located in the non-template strand of the double-stranded DNA molecule ~10 nucleotides upstream of the transcription start site. Here, we explain the mechanism by which the phage AR9 non-virion RNAP (nvRNAP), a bacterial RNAP homolog, recognizes the −10 element of its deoxyuridine-containing promoter in the template strand. The AR9 sigma-like subunit, the nvRNAP enzyme core, and the template strand together form two nucleotide base-accepting pockets whose shapes dictate the requirement for the conserved deoxyuridines. A single amino acid substitution in the AR9 sigma-like subunit allows one of these pockets to accept a thymine thus expanding the promoter consensus. Our work demonstrates the extent to which viruses can evolve host-derived multisubunit enzymes to make transcription of their own genes independent of the host.
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UR - http://www.scopus.com/inward/citedby.url?scp=85132292010&partnerID=8YFLogxK
U2 - 10.1038/s41467-022-31214-6
DO - 10.1038/s41467-022-31214-6
M3 - Article
C2 - 35725571
AN - SCOPUS:85132292010
SN - 2041-1723
VL - 13
JO - Nature Communications
JF - Nature Communications
IS - 1
M1 - 3526
ER -