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. 1998 Jul 15;17(14):4101–4113. doi: 10.1093/emboj/17.14.4101

Structure of T7 RNA polymerase complexed to the transcriptional inhibitor T7 lysozyme.

D Jeruzalmi 1, T A Steitz 1
PMCID: PMC1170743  PMID: 9670025

Abstract

The T7 RNA polymerase-T7 lysozyme complex regulates phage gene expression during infection of Escherichia coli. The 2.8 A crystal structure of the complex reveals that lysozyme binds at a site remote from the polymerase active site, suggesting an indirect mechanism of inhibition. Comparison of the T7 RNA polymerase structure with that of the homologous pol I family of DNA polymerases reveals identities in the catalytic site but also differences specific to RNA polymerase function. The structure of T7 RNA polymerase presented here differs significantly from a previously published structure. Sequence similarities between phage RNA polymerases and those from mitochondria and chloroplasts, when interpreted in the context of our revised model of T7 RNA polymerase, suggest a conserved fold.

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Selected References

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  1. Abdel-Meguid S. S., Jeruzalmi D., Sanderson M. R. Preliminary characterization of crystals. Methods Mol Biol. 1996;56:55–86. doi: 10.1385/0-89603-259-0:55. [DOI] [PubMed] [Google Scholar]
  2. Adams P. D., Pannu N. S., Read R. J., Brünger A. T. Cross-validated maximum likelihood enhances crystallographic simulated annealing refinement. Proc Natl Acad Sci U S A. 1997 May 13;94(10):5018–5023. doi: 10.1073/pnas.94.10.5018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arnold E., Ding J., Hughes S. H., Hostomsky Z. Structures of DNA and RNA polymerases and their interactions with nucleic acid substrates. Curr Opin Struct Biol. 1995 Feb;5(1):27–38. doi: 10.1016/0959-440x(95)80006-m. [DOI] [PubMed] [Google Scholar]
  4. Barton G. J. ALSCRIPT: a tool to format multiple sequence alignments. Protein Eng. 1993 Jan;6(1):37–40. doi: 10.1093/protein/6.1.37. [DOI] [PubMed] [Google Scholar]
  5. Bonner G., Lafer E. M., Sousa R. Characterization of a set of T7 RNA polymerase active site mutants. J Biol Chem. 1994 Oct 7;269(40):25120–25128. [PubMed] [Google Scholar]
  6. Bonner G., Lafer E. M., Sousa R. The thumb subdomain of T7 RNA polymerase functions to stabilize the ternary complex during processive transcription. J Biol Chem. 1994 Oct 7;269(40):25129–25136. [PubMed] [Google Scholar]
  7. Bonner G., Patra D., Lafer E. M., Sousa R. Mutations in T7 RNA polymerase that support the proposal for a common polymerase active site structure. EMBO J. 1992 Oct;11(10):3767–3775. doi: 10.1002/j.1460-2075.1992.tb05462.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brautigam C. A., Steitz T. A. Structural and functional insights provided by crystal structures of DNA polymerases and their substrate complexes. Curr Opin Struct Biol. 1998 Feb;8(1):54–63. doi: 10.1016/s0959-440x(98)80010-9. [DOI] [PubMed] [Google Scholar]
  9. Cheng X., Zhang X., Pflugrath J. W., Studier F. W. The structure of bacteriophage T7 lysozyme, a zinc amidase and an inhibitor of T7 RNA polymerase. Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):4034–4038. doi: 10.1073/pnas.91.9.4034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Delarue M., Poch O., Tordo N., Moras D., Argos P. An attempt to unify the structure of polymerases. Protein Eng. 1990 May;3(6):461–467. doi: 10.1093/protein/3.6.461. [DOI] [PubMed] [Google Scholar]
  11. Doublié S., Tabor S., Long A. M., Richardson C. C., Ellenberger T. Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 A resolution. Nature. 1998 Jan 15;391(6664):251–258. doi: 10.1038/34593. [DOI] [PubMed] [Google Scholar]
  12. Eom S. H., Wang J., Steitz T. A. Structure of Taq polymerase with DNA at the polymerase active site. Nature. 1996 Jul 18;382(6588):278–281. doi: 10.1038/382278a0. [DOI] [PubMed] [Google Scholar]
  13. Erie D. A., Yager T. D., von Hippel P. H. The single-nucleotide addition cycle in transcription: a biophysical and biochemical perspective. Annu Rev Biophys Biomol Struct. 1992;21:379–415. doi: 10.1146/annurev.bb.21.060192.002115. [DOI] [PubMed] [Google Scholar]
  14. Fleischmann R. D., Adams M. D., White O., Clayton R. A., Kirkness E. F., Kerlavage A. R., Bult C. J., Tomb J. F., Dougherty B. A., Merrick J. M. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science. 1995 Jul 28;269(5223):496–512. doi: 10.1126/science.7542800. [DOI] [PubMed] [Google Scholar]
  15. Gao G., Orlova M., Georgiadis M. M., Hendrickson W. A., Goff S. P. Conferring RNA polymerase activity to a DNA polymerase: a single residue in reverse transcriptase controls substrate selection. Proc Natl Acad Sci U S A. 1997 Jan 21;94(2):407–411. doi: 10.1073/pnas.94.2.407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gardner L. P., Mookhtiar K. A., Coleman J. E. Initiation, elongation, and processivity of carboxyl-terminal mutants of T7 RNA polymerase. Biochemistry. 1997 Mar 11;36(10):2908–2918. doi: 10.1021/bi962397i. [DOI] [PubMed] [Google Scholar]
  17. Gross L., Chen W. J., McAllister W. T. Characterization of bacteriophage T7 RNA polymerase by linker insertion mutagenesis. J Mol Biol. 1992 Nov 20;228(2):488–505. doi: 10.1016/0022-2836(92)90837-a. [DOI] [PubMed] [Google Scholar]
  18. He B., Rong M., Durbin R. K., McAllister W. T. A mutant T7 RNA polymerase that is defective in RNA binding and blocked in the early stages of transcription. J Mol Biol. 1997 Jan 24;265(3):275–288. doi: 10.1006/jmbi.1996.0741. [DOI] [PubMed] [Google Scholar]
  19. Hedtke B., Börner T., Weihe A. Mitochondrial and chloroplast phage-type RNA polymerases in Arabidopsis. Science. 1997 Aug 8;277(5327):809–811. doi: 10.1126/science.277.5327.809. [DOI] [PubMed] [Google Scholar]
  20. Holm L., Sander C. Searching protein structure databases has come of age. Proteins. 1994 Jul;19(3):165–173. doi: 10.1002/prot.340190302. [DOI] [PubMed] [Google Scholar]
  21. Ikeda R. A., Bailey P. A. Inhibition of T7 RNA polymerase by T7 lysozyme in vitro. J Biol Chem. 1992 Oct 5;267(28):20153–20158. [PubMed] [Google Scholar]
  22. Ikeda R. A., Richardson C. C. Enzymatic properties of a proteolytically nicked RNA polymerase of bacteriophage T7. J Biol Chem. 1987 Mar 15;262(8):3790–3799. [PubMed] [Google Scholar]
  23. Ikeda R. A., Richardson C. C. Interactions of the RNA polymerase of bacteriophage T7 with its promoter during binding and initiation of transcription. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3614–3618. doi: 10.1073/pnas.83.11.3614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Inouye M., Arnheim N., Sternglanz R. Bacteriophage T7 lysozyme is an N-acetylmuramyl-L-alanine amidase. J Biol Chem. 1973 Oct 25;248(20):7247–7252. [PubMed] [Google Scholar]
  25. Jacobo-Molina A., Ding J., Nanni R. G., Clark A. D., Jr, Lu X., Tantillo C., Williams R. L., Kamer G., Ferris A. L., Clark P. Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded DNA at 3.0 A resolution shows bent DNA. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6320–6324. doi: 10.1073/pnas.90.13.6320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Jeruzalmi D., Steitz T. A. Use of organic cosmotropic solutes to crystallize flexible proteins: application to T7 RNA polymerase and its complex with the inhibitor T7 lysozyme. J Mol Biol. 1997 Dec 19;274(5):748–756. doi: 10.1006/jmbi.1997.1366. [DOI] [PubMed] [Google Scholar]
  27. Jones T. A., Zou J. Y., Cowan S. W., Kjeldgaard M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A. 1991 Mar 1;47(Pt 2):110–119. doi: 10.1107/s0108767390010224. [DOI] [PubMed] [Google Scholar]
  28. Joyce C. M. Choosing the right sugar: how polymerases select a nucleotide substrate. Proc Natl Acad Sci U S A. 1997 Mar 4;94(5):1619–1622. doi: 10.1073/pnas.94.5.1619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Joyce C. M., Steitz T. A. Function and structure relationships in DNA polymerases. Annu Rev Biochem. 1994;63:777–822. doi: 10.1146/annurev.bi.63.070194.004021. [DOI] [PubMed] [Google Scholar]
  30. Kiefer J. R., Mao C., Braman J. C., Beese L. S. Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. Nature. 1998 Jan 15;391(6664):304–307. doi: 10.1038/34693. [DOI] [PubMed] [Google Scholar]
  31. Kim Y., Eom S. H., Wang J., Lee D. S., Suh S. W., Steitz T. A. Crystal structure of Thermus aquaticus DNA polymerase. Nature. 1995 Aug 17;376(6541):612–616. doi: 10.1038/376612a0. [DOI] [PubMed] [Google Scholar]
  32. Kleywegt G. J., Jones T. A. Efficient rebuilding of protein structures. Acta Crystallogr D Biol Crystallogr. 1996 Jul 1;52(Pt 4):829–832. doi: 10.1107/S0907444996001783. [DOI] [PubMed] [Google Scholar]
  33. Kleywegt G. J., Read R. J. Not your average density. Structure. 1997 Dec 15;5(12):1557–1569. doi: 10.1016/s0969-2126(97)00305-5. [DOI] [PubMed] [Google Scholar]
  34. Kohlstaedt L. A., Wang J., Friedman J. M., Rice P. A., Steitz T. A. Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science. 1992 Jun 26;256(5065):1783–1790. doi: 10.1126/science.1377403. [DOI] [PubMed] [Google Scholar]
  35. Kostyuk D. A., Dragan S. M., Lyakhov D. L., Rechinsky V. O., Tunitskaya V. L., Chernov B. K., Kochetkov S. N. Mutants of T7 RNA polymerase that are able to synthesize both RNA and DNA. FEBS Lett. 1995 Aug 7;369(2-3):165–168. doi: 10.1016/0014-5793(95)00732-o. [DOI] [PubMed] [Google Scholar]
  36. Kumar A., Patel S. S. Inhibition of T7 RNA polymerase: transcription initiation and transition from initiation to elongation are inhibited by T7 lysozyme via a ternary complex with RNA polymerase and promoter DNA. Biochemistry. 1997 Nov 11;36(45):13954–13962. doi: 10.1021/bi971432y. [DOI] [PubMed] [Google Scholar]
  37. Ling M. L., Risman S. S., Klement J. F., McGraw N., McAllister W. T. Abortive initiation by bacteriophage T3 and T7 RNA polymerases under conditions of limiting substrate. Nucleic Acids Res. 1989 Feb 25;17(4):1605–1618. doi: 10.1093/nar/17.4.1605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Livingstone C. D., Barton G. J. Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation. Comput Appl Biosci. 1993 Dec;9(6):745–756. doi: 10.1093/bioinformatics/9.6.745. [DOI] [PubMed] [Google Scholar]
  39. Lyakhov D. L., He B., Zhang X., Studier F. W., Dunn J. J., McAllister W. T. Mutant bacteriophage T7 RNA polymerases with altered termination properties. J Mol Biol. 1997 May 30;269(1):28–40. doi: 10.1006/jmbi.1997.1015. [DOI] [PubMed] [Google Scholar]
  40. Martin C. T., Muller D. K., Coleman J. E. Processivity in early stages of transcription by T7 RNA polymerase. Biochemistry. 1988 May 31;27(11):3966–3974. doi: 10.1021/bi00411a012. [DOI] [PubMed] [Google Scholar]
  41. McAllister W. T., Morris C., Rosenberg A. H., Studier F. W. Utilization of bacteriophage T7 late promoters in recombinant plasmids during infection. J Mol Biol. 1981 Dec 15;153(3):527–544. doi: 10.1016/0022-2836(81)90406-x. [DOI] [PubMed] [Google Scholar]
  42. McAllister W. T., Wu H. L. Regulation of transcription of the late genes of bacteriophage T7. Proc Natl Acad Sci U S A. 1978 Feb;75(2):804–808. doi: 10.1073/pnas.75.2.804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Minnick D. T., Astatke M., Joyce C. M., Kunkel T. A. A thumb subdomain mutant of the large fragment of Escherichia coli DNA polymerase I with reduced DNA binding affinity, processivity, and frameshift fidelity. J Biol Chem. 1996 Oct 4;271(40):24954–24961. doi: 10.1074/jbc.271.40.24954. [DOI] [PubMed] [Google Scholar]
  44. Moffatt B. A., Studier F. W. T7 lysozyme inhibits transcription by T7 RNA polymerase. Cell. 1987 Apr 24;49(2):221–227. doi: 10.1016/0092-8674(87)90563-0. [DOI] [PubMed] [Google Scholar]
  45. Mookhtiar K. A., Peluso P. S., Muller D. K., Dunn J. J., Coleman J. E. Processivity of T7 RNA polymerase requires the C-terminal Phe882-Ala883-COO- or "foot". Biochemistry. 1991 Jun 25;30(25):6305–6313. doi: 10.1021/bi00239a032. [DOI] [PubMed] [Google Scholar]
  46. Muller D. K., Martin C. T., Coleman J. E. Processivity of proteolytically modified forms of T7 RNA polymerase. Biochemistry. 1988 Jul 26;27(15):5763–5771. doi: 10.1021/bi00415a055. [DOI] [PubMed] [Google Scholar]
  47. Ollis D. L., Brick P., Hamlin R., Xuong N. G., Steitz T. A. Structure of large fragment of Escherichia coli DNA polymerase I complexed with dTMP. 1985 Feb 28-Mar 6Nature. 313(6005):762–766. doi: 10.1038/313762a0. [DOI] [PubMed] [Google Scholar]
  48. Osumi-Davis P. A., de Aguilera M. C., Woody R. W., Woody A. Y. Asp537, Asp812 are essential and Lys631, His811 are catalytically significant in bacteriophage T7 RNA polymerase activity. J Mol Biol. 1992 Jul 5;226(1):37–45. doi: 10.1016/0022-2836(92)90122-z. [DOI] [PubMed] [Google Scholar]
  49. Patra D., Lafer E. M., Sousa R. Isolation and characterization of mutant bacteriophage T7 RNA polymerases. J Mol Biol. 1992 Mar 20;224(2):307–318. doi: 10.1016/0022-2836(92)90996-w. [DOI] [PubMed] [Google Scholar]
  50. Pelletier H., Sawaya M. R., Kumar A., Wilson S. H., Kraut J. Structures of ternary complexes of rat DNA polymerase beta, a DNA template-primer, and ddCTP. Science. 1994 Jun 24;264(5167):1891–1903. [PubMed] [Google Scholar]
  51. Raskin C. A., Diaz G., Joho K., McAllister W. T. Substitution of a single bacteriophage T3 residue in bacteriophage T7 RNA polymerase at position 748 results in a switch in promoter specificity. J Mol Biol. 1992 Nov 20;228(2):506–515. doi: 10.1016/0022-2836(92)90838-b. [DOI] [PubMed] [Google Scholar]
  52. Rechinsky V. O., Chernov B. K., Dragan S. M., Kostyuk D. A., Tunitskaya V. L., Kochetkov S. N. Targeted mutagenesis identifies Asp-569 as a catalytically critical residue in T7 RNA polymerase. Mol Gen Genet. 1995 Apr 10;247(1):110–113. doi: 10.1007/BF00425827. [DOI] [PubMed] [Google Scholar]
  53. Rechinsky V. O., Tunitskaya V. L., Dragan S. M., Kostyuk D. A., Kochetkov S. N. Tyr-571 is involved in the T7 RNA polymerase binding to its promoter. FEBS Lett. 1993 Mar 29;320(1):9–12. doi: 10.1016/0014-5793(93)81646-h. [DOI] [PubMed] [Google Scholar]
  54. Reeder Franziska, Kozelka Jirí, Chottard J. C. Triammineplatinum(II) Coordinated to a Guanine Does Not Prevent Platination of an Adjacent Guanine in Single-Stranded Oligonucleotides. Inorg Chem. 1996 Feb 28;35(5):1413–1415. doi: 10.1021/ic951135m. [DOI] [PubMed] [Google Scholar]
  55. Sastry S. S., Hearst J. E. Studies on the interaction of T7 RNA polymerase with a DNA template containing a site-specifically placed psoralen cross-link. I. Characterization of elongation complexes. J Mol Biol. 1991 Oct 20;221(4):1091–1110. [PubMed] [Google Scholar]
  56. Sousa R., Chung Y. J., Rose J. P., Wang B. C. Crystal structure of bacteriophage T7 RNA polymerase at 3.3 A resolution. Nature. 1993 Aug 12;364(6438):593–599. doi: 10.1038/364593a0. [DOI] [PubMed] [Google Scholar]
  57. Sousa R., Patra D., Lafer E. M. Model for the mechanism of bacteriophage T7 RNAP transcription initiation and termination. J Mol Biol. 1992 Mar 20;224(2):319–334. doi: 10.1016/0022-2836(92)90997-x. [DOI] [PubMed] [Google Scholar]
  58. Sousa R., Rose J., Wang B. C. The thumb's knuckle. Flexibility in the thumb subdomain of T7 RNA polymerase is revealed by the structure of a chimeric T7/T3 RNA polymerase. J Mol Biol. 1994 Nov 18;244(1):6–12. doi: 10.1006/jmbi.1994.1699. [DOI] [PubMed] [Google Scholar]
  59. Steitz T. A., Smerdon S. J., Jäger J., Joyce C. M. A unified polymerase mechanism for nonhomologous DNA and RNA polymerases. Science. 1994 Dec 23;266(5193):2022–2025. doi: 10.1126/science.7528445. [DOI] [PubMed] [Google Scholar]
  60. Studier F. W. Bacteriophage T7. Science. 1972 Apr 28;176(4033):367–376. doi: 10.1126/science.176.4033.367. [DOI] [PubMed] [Google Scholar]
  61. Studier F. W., Dunn J. J. Organization and expression of bacteriophage T7 DNA. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 2):999–1007. doi: 10.1101/sqb.1983.047.01.114. [DOI] [PubMed] [Google Scholar]
  62. Tjian R. The biochemistry of transcription in eukaryotes: a paradigm for multisubunit regulatory complexes. Philos Trans R Soc Lond B Biol Sci. 1996 Apr 29;351(1339):491–499. doi: 10.1098/rstb.1996.0047. [DOI] [PubMed] [Google Scholar]
  63. Ujvári A., Martin C. T. Identification of a minimal binding element within the T7 RNA polymerase promoter. J Mol Biol. 1997 Nov 7;273(4):775–781. doi: 10.1006/jmbi.1997.1350. [DOI] [PubMed] [Google Scholar]
  64. Wang J., Sattar A. K., Wang C. C., Karam J. D., Konigsberg W. H., Steitz T. A. Crystal structure of a pol alpha family replication DNA polymerase from bacteriophage RB69. Cell. 1997 Jun 27;89(7):1087–1099. doi: 10.1016/s0092-8674(00)80296-2. [DOI] [PubMed] [Google Scholar]
  65. Zhang X., Studier F. W. Mechanism of inhibition of bacteriophage T7 RNA polymerase by T7 lysozyme. J Mol Biol. 1997 May 30;269(1):10–27. doi: 10.1006/jmbi.1997.1016. [DOI] [PubMed] [Google Scholar]
  66. von Hippel P. H., Bear D. G., Morgan W. D., McSwiggen J. A. Protein-nucleic acid interactions in transcription: a molecular analysis. Annu Rev Biochem. 1984;53:389–446. doi: 10.1146/annurev.bi.53.070184.002133. [DOI] [PubMed] [Google Scholar]

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