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. 1994 Mar;176(5):1394–1404. doi: 10.1128/jb.176.5.1394-1404.1994

Escherichia coli-Salmonella typhimurium hybrid nusA genes: identification of a short motif required for action of the lambda N transcription antitermination protein.

M G Craven 1, A E Granston 1, A T Schauer 1, C Zheng 1, T A Gray 1, D I Friedman 1
PMCID: PMC205205  PMID: 8113180

Abstract

The Escherichia coli nusA gene, nusAEc, encodes an essential protein that influences transcription elongation. Derivatives of E. coli in which the Salmonella typhimurium nusA gene, nusASt, has replaced nusAEc are viable. Thus, NusASt can substitute for NusAEc in supporting essential bacterial activities. However, hybrid E. coli strains with the nusASt substitution do not effectively support transcription antitermination mediated by the N gene product of phage lambda. We report the DNA sequence of nusASt, showing that the derived amino acid sequence is 95% identical to the derived amino acid sequence of nusAEc. The alignment of the amino acid sequences reveals scattered single amino acid differences and one region of significant heterogeneity. In this region, called 449, NusAEc has four amino acids and NusASt has nine amino acids. Functional studies of hybrid nusA genes, constructed from nusAEc and nusASt, show that the 449 region of the NusAEc protein is important for lambda N-mediated transcription antitermination. A hybrid that has a substitution of the four E. coli codons for the nine S. typhimurium codons, but is otherwise nusASt, supports the action of the N antitermination protein. The 449 region and, presumably, adjacent sequences appear to compose a functional domain of NusAEc important for the action of the N transcription antitermination protein of phage lambda.

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Selected References

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  1. Barik S., Ghosh B., Whalen W., Lazinski D., Das A. An antitermination protein engages the elongating transcription apparatus at a promoter-proximal recognition site. Cell. 1987 Sep 11;50(6):885–899. doi: 10.1016/0092-8674(87)90515-0. [DOI] [PubMed] [Google Scholar]
  2. Baron L. S., Penido E., Ryman I. R., Falkow S. Behavior of coliphage lambda in hybrids between Escherichia coli and Salmonella. J Bacteriol. 1970 Apr;102(1):221–233. doi: 10.1128/jb.102.1.221-233.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bear S. E., Court D. L., Friedman D. I. An accessory role for Escherichia coli integration host factor: characterization of a lambda mutant dependent upon integration host factor for DNA packaging. J Virol. 1984 Dec;52(3):966–972. doi: 10.1128/jvi.52.3.966-972.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bolivar F. Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique Eco RI sites for selection of Eco RI generated recombinant DNA molecules. Gene. 1978 Oct;4(2):121–136. doi: 10.1016/0378-1119(78)90025-2. [DOI] [PubMed] [Google Scholar]
  5. Calnan B. J., Tidor B., Biancalana S., Hudson D., Frankel A. D. Arginine-mediated RNA recognition: the arginine fork. Science. 1991 May 24;252(5009):1167–1171. doi: 10.1126/science.252.5009.1167. [DOI] [PubMed] [Google Scholar]
  6. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cheng S. W., Lynch E. C., Leason K. R., Court D. L., Shapiro B. A., Friedman D. I. Functional importance of sequence in the stem-loop of a transcription terminator. Science. 1991 Nov 22;254(5035):1205–1207. doi: 10.1126/science.1835546. [DOI] [PubMed] [Google Scholar]
  8. Costantino N., Zuber M., Court D. Analysis of mutations in the ninR region of bacteriophage lambda that bypass a requirement for lambda N antitermination. J Bacteriol. 1990 Aug;172(8):4610–4615. doi: 10.1128/jb.172.8.4610-4615.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Court D., Brady C., Rosenberg M., Wulff D. L., Behr M., Mahoney M., Izumi S. U. Control of transcription termination: a rho-dependent termination site in bacteriophage lambda. J Mol Biol. 1980 Apr;138(2):231–254. doi: 10.1016/0022-2836(80)90285-5. [DOI] [PubMed] [Google Scholar]
  10. Court D., Sato K. Studies of novel transducing variants of lambda: dispensability of genes N and Q. Virology. 1969 Oct;39(2):348–352. doi: 10.1016/0042-6822(69)90060-9. [DOI] [PubMed] [Google Scholar]
  11. Craven M. G., Friedman D. I. Analysis of the Escherichia coli nusA10(Cs) allele: relating nucleotide changes to phenotypes. J Bacteriol. 1991 Feb;173(4):1485–1491. doi: 10.1128/jb.173.4.1485-1491.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cryptic initiation sequence revealed. Nature. 1990 Feb 1;343(6257):417–418. doi: 10.1038/343417b0. [DOI] [PubMed] [Google Scholar]
  13. Das A. How the phage lambda N gene product suppresses transcription termination: communication of RNA polymerase with regulatory proteins mediated by signals in nascent RNA. J Bacteriol. 1992 Nov;174(21):6711–6716. doi: 10.1128/jb.174.21.6711-6716.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Das A., Wolska K. Transcription antitermination in vitro by lambda N gene product: requirement for a phage nut site and the products of host nusA, nusB, and nusE genes. Cell. 1984 Aug;38(1):165–173. doi: 10.1016/0092-8674(84)90537-3. [DOI] [PubMed] [Google Scholar]
  15. Drahos D., Szybalski W. Antitermination and termination functions of the cloned nutL, N, and tL1 modules of coliphage lambda. Gene. 1981 Dec;16(1-3):261–274. doi: 10.1016/0378-1119(81)90082-2. [DOI] [PubMed] [Google Scholar]
  16. Farnham P. J., Greenblatt J., Platt T. Effects of NusA protein on transcription termination in the tryptophan operon of Escherichia coli. Cell. 1982 Jul;29(3):945–951. doi: 10.1016/0092-8674(82)90457-3. [DOI] [PubMed] [Google Scholar]
  17. Faus I., Chen C. Y., Richardson J. P. Sequences in the 5' proximal segment of the paused transcript affect NusA-mediated enhancement of transcriptional pausing. J Biol Chem. 1988 Aug 5;263(22):10830–10835. [PubMed] [Google Scholar]
  18. Franklin N. C. Conservation of genome form but not sequence in the transcription antitermination determinants of bacteriophages lambda, phi 21 and P22. J Mol Biol. 1985 Jan 5;181(1):75–84. doi: 10.1016/0022-2836(85)90325-0. [DOI] [PubMed] [Google Scholar]
  19. Friedman D. I., Baron L. S. Genetic characterization of a bacterial locus involved in the activity of the N function of phage lambda. Virology. 1974 Mar;58(1):141–148. doi: 10.1016/0042-6822(74)90149-4. [DOI] [PubMed] [Google Scholar]
  20. Friedman D. I., Imperiale M. J., Adhya S. L. RNA 3' end formation in the control of gene expression. Annu Rev Genet. 1987;21:453–488. doi: 10.1146/annurev.ge.21.120187.002321. [DOI] [PubMed] [Google Scholar]
  21. Friedman D. I., Jolly C. T., Mural R. J. Interference with the expression of the N gene function of phage lambda in a mutant of Escherichia coli. Virology. 1973 Jan;51(1):216–226. doi: 10.1016/0042-6822(73)90381-4. [DOI] [PubMed] [Google Scholar]
  22. Friedman D. I., Olson E. R. Evidence that a nucleotide sequence, "boxA," is involved in the action of the NusA protein. Cell. 1983 Aug;34(1):143–149. doi: 10.1016/0092-8674(83)90144-7. [DOI] [PubMed] [Google Scholar]
  23. Friedman D. I., Olson E. R., Georgopoulos C., Tilly K., Herskowitz I., Banuett F. Interactions of bacteriophage and host macromolecules in the growth of bacteriophage lambda. Microbiol Rev. 1984 Dec;48(4):299–325. doi: 10.1128/mr.48.4.299-325.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Friedman D. I., Olson E. R., Johnson L. L., Alessi D., Craven M. G. Transcription-dependent competition for a host factor: the function and optimal sequence of the phage lambda boxA transcription antitermination signal. Genes Dev. 1990 Dec;4(12A):2210–2222. doi: 10.1101/gad.4.12a.2210. [DOI] [PubMed] [Google Scholar]
  25. Friedman D. I., Schauer A. T., Baumann M. R., Baron L. S., Adhya S. L. Evidence that ribosomal protein S10 participates in control of transcription termination. Proc Natl Acad Sci U S A. 1981 Feb;78(2):1115–1118. doi: 10.1073/pnas.78.2.1115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Friedman D. I., Wilgus G. S., Mural R. J. Gene N regulator function of phage lambda immun21: evidence that a site of N action differs from a site of N recognition. J Mol Biol. 1973 Dec 25;81(4):505–516. doi: 10.1016/0022-2836(73)90519-6. [DOI] [PubMed] [Google Scholar]
  27. Gill S. C., Weitzel S. E., von Hippel P. H. Escherichia coli sigma 70 and NusA proteins. I. Binding interactions with core RNA polymerase in solution and within the transcription complex. J Mol Biol. 1991 Jul 20;220(2):307–324. doi: 10.1016/0022-2836(91)90015-x. [DOI] [PubMed] [Google Scholar]
  28. Goda Y., Greenblatt J. Efficient modification of E. coli RNA polymerase in vitro by the N gene transcription antitermination protein of bacteriophage lambda. Nucleic Acids Res. 1985 Apr 11;13(7):2569–2582. doi: 10.1093/nar/13.7.2569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Granston A. E., Thompson D. L., Friedman D. I. Identification of a second promoter for the metY-nusA-infB operon of Escherichia coli. J Bacteriol. 1990 May;172(5):2336–2342. doi: 10.1128/jb.172.5.2336-2342.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Greenblatt J., Li J. Interaction of the sigma factor and the nusA gene protein of E. coli with RNA polymerase in the initiation-termination cycle of transcription. Cell. 1981 May;24(2):421–428. doi: 10.1016/0092-8674(81)90332-9. [DOI] [PubMed] [Google Scholar]
  31. Greenblatt J., Li J. Properties of the N gene transcription antitermination protein of bacteriophage lambda. J Biol Chem. 1982 Jan 10;257(1):362–365. [PubMed] [Google Scholar]
  32. Greenblatt J., Li J. The nusA gene protein of Escherichia coli. Its identification and a demonstration that it interacts with the gene N transcription anti-termination protein of bacteriophage lambda. J Mol Biol. 1981 Mar 25;147(1):11–23. doi: 10.1016/0022-2836(81)90076-0. [DOI] [PubMed] [Google Scholar]
  33. Greenblatt J., McLimont M., Hanly S. Termination of transcription by nusA gene protein of Escherichia coli. Nature. 1981 Jul 16;292(5820):215–220. doi: 10.1038/292215a0. [DOI] [PubMed] [Google Scholar]
  34. Hilliker S., Botstein D. Specificity of genetic elements controlling regulation of early functions in temperate bacteriophages. J Mol Biol. 1976 Sep 25;106(3):537–566. doi: 10.1016/0022-2836(76)90251-5. [DOI] [PubMed] [Google Scholar]
  35. Horton R. M., Hunt H. D., Ho S. N., Pullen J. K., Pease L. R. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene. 1989 Apr 15;77(1):61–68. doi: 10.1016/0378-1119(89)90359-4. [DOI] [PubMed] [Google Scholar]
  36. Horwitz R. J., Li J., Greenblatt J. An elongation control particle containing the N gene transcriptional antitermination protein of bacteriophage lambda. Cell. 1987 Nov 20;51(4):631–641. doi: 10.1016/0092-8674(87)90132-2. [DOI] [PubMed] [Google Scholar]
  37. Ishii S., Ihara M., Maekawa T., Nakamura Y., Uchida H., Imamoto F. The nucleotide sequence of the cloned nusA gene and its flanking region of Escherichia coli. Nucleic Acids Res. 1984 Apr 11;12(7):3333–3342. doi: 10.1093/nar/12.7.3333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Ito K., Egawa K., Nakamura Y. Genetic interaction between the beta' subunit of RNA polymerase and the arginine-rich domain of Escherichia coli nusA protein. J Bacteriol. 1991 Feb;173(4):1492–1501. doi: 10.1128/jb.173.4.1492-1501.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Jin D. J., Cashel M., Friedman D. I., Nakamura Y., Walter W. A., Gross C. A. Effects of rifampicin resistant rpoB mutations on antitermination and interaction with nusA in Escherichia coli. J Mol Biol. 1988 Nov 20;204(2):247–261. doi: 10.1016/0022-2836(88)90573-6. [DOI] [PubMed] [Google Scholar]
  40. Kingston R. E., Chamberlin M. J. Pausing and attenuation of in vitro transcription in the rrnB operon of E. coli. Cell. 1981 Dec;27(3 Pt 2):523–531. doi: 10.1016/0092-8674(81)90394-9. [DOI] [PubMed] [Google Scholar]
  41. Kröger M., Hobom G. A chain of interlinked genes in the ninR region of bacteriophage lambda. Gene. 1982 Nov;20(1):25–38. doi: 10.1016/0378-1119(82)90084-1. [DOI] [PubMed] [Google Scholar]
  42. Kung H., Spears C., Weissbach H. Purification and properties of a soluble factor required for the deoxyribonucleic acid-directed in vitro synthesis of beta-galactosidase. J Biol Chem. 1975 Feb 25;250(4):1556–1562. [PubMed] [Google Scholar]
  43. Lau L. F., Roberts J. W., Wu R. RNA polymerase pausing and transcript release at the lambda tR1 terminator in vitro. J Biol Chem. 1983 Aug 10;258(15):9391–9397. [PubMed] [Google Scholar]
  44. Lazinski D., Grzadzielska E., Das A. Sequence-specific recognition of RNA hairpins by bacteriophage antiterminators requires a conserved arginine-rich motif. Cell. 1989 Oct 6;59(1):207–218. doi: 10.1016/0092-8674(89)90882-9. [DOI] [PubMed] [Google Scholar]
  45. Leason K. R., Friedman D. I. Analysis of transcription termination signals in the nin region of bacteriophage lambda: the roc deletion. J Bacteriol. 1988 Nov;170(11):5051–5058. doi: 10.1128/jb.170.11.5051-5058.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Mason S. W., Greenblatt J. Assembly of transcription elongation complexes containing the N protein of phage lambda and the Escherichia coli elongation factors NusA, NusB, NusG, and S10. Genes Dev. 1991 Aug;5(8):1504–1512. doi: 10.1101/gad.5.8.1504. [DOI] [PubMed] [Google Scholar]
  47. Mason S. W., Li J., Greenblatt J. Host factor requirements for processive antitermination of transcription and suppression of pausing by the N protein of bacteriophage lambda. J Biol Chem. 1992 Sep 25;267(27):19418–19426. [PubMed] [Google Scholar]
  48. Miller H. I., Friedman D. I. An E. coli gene product required for lambda site-specific recombination. Cell. 1980 Jul;20(3):711–719. doi: 10.1016/0092-8674(80)90317-7. [DOI] [PubMed] [Google Scholar]
  49. Miller H. I., Mozola M. A., Friedman D. I. int-h: An int mutation of phage lambda that enhances site-specific recombination. Cell. 1980 Jul;20(3):721–729. doi: 10.1016/0092-8674(80)90318-9. [DOI] [PubMed] [Google Scholar]
  50. Mozola M. A., Friedman D. I., Crawford C. L., Wulff D. L., Shimatake H., Rosenberg M. Mutations reducing the activity of c17, a promoter of phage lambda formed by a tandem duplication. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1122–1125. doi: 10.1073/pnas.76.3.1122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Nakamura Y., Mizusawa S., Court D. L., Tsugawa A. Regulatory defects of a conditionally lethal nusAts mutant of Escherichia coli. Positive and negative modulator roles of NusA protein in vivo. J Mol Biol. 1986 May 5;189(1):103–111. doi: 10.1016/0022-2836(86)90384-0. [DOI] [PubMed] [Google Scholar]
  52. Nakamura Y., Mizusawa S. In vivo evidence that the nusA and infB genes of E. coli are part of the same multi-gene operon which encodes at least four proteins. EMBO J. 1985 Feb;4(2):527–532. doi: 10.1002/j.1460-2075.1985.tb03660.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Nakamura Y., Mizusawa S., Tsugawa A., Imai M. Conditionally lethal nusAts mutation of Escherichia coli reduces transcription termination but does not affect antitermination of bacteriophage lambda. Mol Gen Genet. 1986 Jul;204(1):24–28. doi: 10.1007/BF00330182. [DOI] [PubMed] [Google Scholar]
  54. Nodwell J. R., Greenblatt J. The nut site of bacteriophage lambda is made of RNA and is bound by transcription antitermination factors on the surface of RNA polymerase. Genes Dev. 1991 Nov;5(11):2141–2151. doi: 10.1101/gad.5.11.2141. [DOI] [PubMed] [Google Scholar]
  55. Reyes O., Gottesman M., Adhya S. Formation of lambda lysogens by IS2 recombination: gal operon--lambda pR promoter fusions. Virology. 1979 Apr 30;94(2):400–408. doi: 10.1016/0042-6822(79)90470-7. [DOI] [PubMed] [Google Scholar]
  56. Riley M., Anilionis A. Evolution of the bacterial genome. Annu Rev Microbiol. 1978;32:519–560. doi: 10.1146/annurev.mi.32.100178.002511. [DOI] [PubMed] [Google Scholar]
  57. Roberts J. W. Phage lambda and the regulation of transcription termination. Cell. 1988 Jan 15;52(1):5–6. doi: 10.1016/0092-8674(88)90523-5. [DOI] [PubMed] [Google Scholar]
  58. Saito M., Tsugawa A., Egawa K., Nakamura Y. Revised sequence of the nusA gene of Escherichia coli and identification of nusA11 (ts) and nusA1 mutations which cause changes in a hydrophobic amino acid cluster. Mol Gen Genet. 1986 Nov;205(2):380–382. doi: 10.1007/BF00430455. [DOI] [PubMed] [Google Scholar]
  59. Salstrom J. S., Szybalski W. Coliphage lambdanutL-: a unique class of mutants defective in the site of gene N product utilization for antitermination of leftward transcription. J Mol Biol. 1978 Sep 5;124(1):195–221. doi: 10.1016/0022-2836(78)90156-0. [DOI] [PubMed] [Google Scholar]
  60. Salstrom J. S., Szybalski W. Transcription termination sites in the major leftward operon of coliphage lambda. Virology. 1978 Jul 15;88(2):252–262. doi: 10.1016/0042-6822(78)90282-9. [DOI] [PubMed] [Google Scholar]
  61. Sanderson K. E. Genetic relatedness in the family Enterobacteriaceae. Annu Rev Microbiol. 1976;30:327–349. doi: 10.1146/annurev.mi.30.100176.001551. [DOI] [PubMed] [Google Scholar]
  62. Schauer A. T., Carver D. L., Bigelow B., Baron L. S., Friedman D. I. lambda N antitermination system: functional analysis of phage interactions with the host NusA protein. J Mol Biol. 1987 Apr 20;194(4):679–690. doi: 10.1016/0022-2836(87)90245-2. [DOI] [PubMed] [Google Scholar]
  63. Schmidt M. C., Chamberlin M. J. Binding of rho factor to Escherichia coli RNA polymerase mediated by nusA protein. J Biol Chem. 1984 Dec 25;259(24):15000–15002. [PubMed] [Google Scholar]
  64. Schmidt M. C., Chamberlin M. J. nusA protein of Escherichia coli is an efficient transcription termination factor for certain terminator sites. J Mol Biol. 1987 Jun 20;195(4):809–818. doi: 10.1016/0022-2836(87)90486-4. [DOI] [PubMed] [Google Scholar]
  65. Sigmund C. D., Morgan E. A. Nus A protein affects transcriptional pausing and termination in vitro by binding to different sites on the transcription complex. Biochemistry. 1988 Jul 26;27(15):5622–5627. doi: 10.1021/bi00415a034. [DOI] [PubMed] [Google Scholar]
  66. Sternberg N. L., Maurer R. Bacteriophage-mediated generalized transduction in Escherichia coli and Salmonella typhimurium. Methods Enzymol. 1991;204:18–43. doi: 10.1016/0076-6879(91)04004-8. [DOI] [PubMed] [Google Scholar]
  67. Tao J., Frankel A. D. Specific binding of arginine to TAR RNA. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2723–2726. doi: 10.1073/pnas.89.7.2723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Tsugawa A., Kurihara T., Zuber M., Court D. L., Nakamura Y. E. coli NusA protein binds in vitro to an RNA sequence immediately upstream of the boxA signal of bacteriophage lambda. EMBO J. 1985 Sep;4(9):2337–2342. doi: 10.1002/j.1460-2075.1985.tb03935.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Tsugawa A., Saito M., Court D. L., Nakamura Y. nusA amber mutation that causes temperature-sensitive growth of Escherichia coli. J Bacteriol. 1988 Feb;170(2):908–915. doi: 10.1128/jb.170.2.908-915.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Ward D. F., Gottesman M. E. The nus mutations affect transcription termination in Escherichia coli. Nature. 1981 Jul 16;292(5820):212–215. doi: 10.1038/292212a0. [DOI] [PubMed] [Google Scholar]
  71. Whalen W., Ghosh B., Das A. NusA protein is necessary and sufficient in vitro for phage lambda N gene product to suppress a rho-independent terminator placed downstream of nutL. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2494–2498. doi: 10.1073/pnas.85.8.2494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  73. Zengel J. M., Lindahl L. Ribosomal protein L4 and transcription factor NusA have separable roles in mediating terminating of transcription within the leader of the S10 operon of Escherichia coli. Genes Dev. 1992 Dec;6(12B):2655–2662. doi: 10.1101/gad.6.12b.2655. [DOI] [PubMed] [Google Scholar]

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