1887

Abstract

Although the attenuator located between the ribosomal protein and RNA polymerase gene domains of the operon has a maximum termination efficiency of 80%, the level of termination is diminished with decreasing transcription frequency. In this report, the use of transcriptional fusions to further investigate the mechanism of transcription-frequency-dependent regulation is described. The termination efficiency of two other weak terminators was assayed over a wide range of transcription frequencies programmed by different strength promoters. The results indicated that a decrease in termination efficiency with decreasing transcription frequency is not an inherent property of weak terminators. Deletion of the 165 bp segment located 439-274 bp upstream of the attenuator abrogated the difference in termination efficiency normally seen between high and low levels of transcription. This suggests that a -acting site located in this upstream region is necessary for transcription-frequency-dependent modulation of the attenuator's function. However, this site apparently works only in combination with the attenuator, since it did not cause transcription-frequency-dependent modulation when placed upstream of two other weak terminators. Analysis of the readthrough frequencies of single or tandem copies of the attenuator indicated that the transcription complexes which pass through the attenuator have not been converted to termination-resistant complexes in a manner analogous to the N-mediated antitermination system of lambda. Finally, an examination of termination efficiency in three mutants suggested that although NusA increases readthrough at the attenuator it is not directly involved in transcription-frequency-dependent modulation.

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1997-11-01
2021-10-20
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References

  1. Appleyard R.K. 1954; Segregation of new lysogenic types during growth of a doubly lysogenic strain derived from Escherichia coli K12.. Genetics 39:440–452
    [Google Scholar]
  2. Brosius J., Dull T.J., Sleater D.D., Noller H.F. 1981; Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. . J Mol Biol 148:107–127
    [Google Scholar]
  3. Cheng S.C., Lynch E.C., Leason K.R., Court D.L., Shapiro B.A., Friedman D.J. 1991; Functional importance of sequence in the stem-loop of a transcriptional terminator.. Science 254:1205–1207
    [Google Scholar]
  4. Clark D.J., Maaloe O. 1967; DNA replication and the division cycle in Escherichia coli. . J Mol Biol 23:99–112
    [Google Scholar]
  5. Craven M.G., Friedman D.I. 1991; Analysis of the Escherichia coli nusA10{Cs) allele: relating nucleotide changes to phenotypes.. J Bacterial 173:1485–1491
    [Google Scholar]
  6. Das A. 1992; 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 174:6711–6716
    [Google Scholar]
  7. Dykxhoorn D.M., St Pierre R., Linn T. 1996; Synthesis of the ? and ? subunits of Escherichia coli RNA polymerase is autogeneously regulated in vivo by both transcriptional and translational mechanisms.. Mol Microbiol 19:483–493
    [Google Scholar]
  8. Engbaek F., Gross C., Burgess R.R. 1976; Quantitation of RNA polymerase subunits in Escherichia coli during exponential growth and after bacteriophage T4 infection.. Mol Gen Genet 143:291–295
    [Google Scholar]
  9. Friedman D.I. 1971; A bacterial mutant affecting lambda development.. In The Bacteriophage Lambda pp. 733–738 Edited by Hershey D. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  10. Friedman D.I. 1988; Regulation of gene expression by termination and antitermination of transcription.. In The Bacterio-phages 2 pp. 263–319 Edited by Calendar R. New York: Plenum;
    [Google Scholar]
  11. Greenblatt J., Nodwell J.R., Mason S.W. 1993; Transcrip-tional antitermination.. Nature 364:401–406
    [Google Scholar]
  12. Hanahan D. 1985; Techniques for transformation of E. coli. . In DNA Cloning 1 pp. 109–135 Edited by Glover D. M. Oxford: IRL Press;
    [Google Scholar]
  13. Hayward R.S., Fyfe S.K. 1978; Oversynthesis and instability of sigma protein in a merodiploid strain of Escherichia coli. . Mol Gen Genet 159:89–99
    [Google Scholar]
  14. von Hippel P.H., Yager T.D. 1992; The elongation-ter-mination decision in transcription.. Science 255:809–812
    [Google Scholar]
  15. Holmes D.S., Quigley M. 1981; A rapid boiling method for the preparation of bacterial plasmids.. Anal Biochem 114:193–197
    [Google Scholar]
  16. Ishihama A. 1993; Protein-protein communication within the transcription apparatus.. J Bacteriol 175:2483–2489
    [Google Scholar]
  17. Iwakura Y., Ito K., Ishihama A. 1974; Biosynthesis of RNA polymerase in Escherichia coli. I. Control of RNA polymerase content at various growth rates.. Mol Gen Genet 133:1–23
    [Google Scholar]
  18. Kawakami K., Saitoh T., Ishihama A. 1979; Biosynthesis of RNA polymerase in Escherichia coli. IX. Growth-dependent variations in the synthesis rate, content and distribution of RNA polymerase.. Mol Gen Genet 174:107–116
    [Google Scholar]
  19. Landick R., Yanofsky C. 1987; Transcription attenuation.. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology pp. 1276–1301 Edited by Neidhardt F. C. others Washington, DC: American Society for Microbiology;
    [Google Scholar]
  20. Linn T., Greenblatt J. 1992; The NusA and NusG proteins of Escherichia coli increase the in vitro readthrough frequency of a transcriptional attenuator preceding the gene for the ? subunit of RNA polymerase.. J Biol Chem 267:1449–1454
    [Google Scholar]
  21. Linn T., St Pierre R. 1990; Improved vector system for constructing transcriptional fusions that ensures independent translation of lacZ. . J Bacteriol 172:1077–1084
    [Google Scholar]
  22. Maniatis T., Fritsch E.F., Sambrook J. 1982 Molecular Cloning: a Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  23. Miller J.H. 1972 Experiments in Molecular Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  24. Mousset S., Thomas R. 1969; Ter, a function which generates the ends of the mature lambda chromosome.. Nature 221:242–244
    [Google Scholar]
  25. Moyle H., Waldburger C., Susskind M.M. 1991; Hierarchies of base pair preferences in the p22 ant promoter.. J Bacteriol 173:1944–1950
    [Google Scholar]
  26. Nakamura Y., Mizusawa S., Court D.L., Tsugawa A. 1986; Regulatory defects of a conditionally lethal nusAts mutant of Escherichia coli. Positive and negative modulator roles of NusA protein in vivo. . J Mol Biol 189:103–111
    [Google Scholar]
  27. Pedersen S., Reeh S.V., Parker J., Watson R.J., Friesen J.D., Fiil N.P. 1976; Analysis of the proteins synthesized in ultraviolet light-irradiated Escherichia coli following infection with the bacteriophage ?drifd18 and ?dfus3.. Mol Gen Genet 144:339–343
    [Google Scholar]
  28. Post L.E., Strycharz G.D., Nomura M., Lewis H., Dennis P.P. 1979; Nucleotide sequence of the ribosomal protein gene cluster adjacent to the gene for RNA polymerase subunit beta in Escherichia coli. . Proc Natl Acad Sci USA 761697–1701
    [Google Scholar]
  29. Railing G., Linn T. 1987; Evidence that Rho and NusA are involved in termination in the rplL-rpoB intercistronic region.. J Bacteriol 169:2277–2280
    [Google Scholar]
  30. Railing G., Bodrug S., Linn T. 1985; Growth rate-dependent regulation of RNA polymerase synthesis in Escherichia coli. . Mol Gen Genet 201:379–386
    [Google Scholar]
  31. Sambrook J., Fritsch E.F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn.. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  32. Santos M.A. 1991; An improved method for the small scale preparation of bacteriophage DNA based on phage precipitation by zinc chloride.. Nucleic Acids Res 19:5442
    [Google Scholar]
  33. Schauer A.T., Carver D.L., Bigelow B., Baron L.S., Friedman D.L>. 1987; Lambda N antitermination system: functional analysis of phage interactions with the host NusA protein.. J Mol Biol 194:679–690
    [Google Scholar]
  34. Schmidt M.C., Chamberlin M.J. 1987; NusA protein of Escherichia coli is an efficient transcription termination factor for certain terminator sites.. J Mol Biol 195:809–818
    [Google Scholar]
  35. Steward K.L., Linn T. 1991; In vivo analysis of overlapping transcription units in the rplKAJLrpoBC ribosomal protein-RNA polymerase gene cluster of Escherichia coli. . J Mol Biol 218:23–31
    [Google Scholar]
  36. Steward K., Linn T. 1992; Transcription frequency modulates the efficiency of an attenuator preceding the rpoBC RNA polymerase genes of Escherichia coli: possible autogenous control.. Nucleic Acids Res 20:4773–4779
    [Google Scholar]
  37. Yager T.D., von Hippel P.H. 1987; Transcript elongation and termination in Escherichia coli. . In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology pp. 1241–1275 Edited by Neidhardt F. C. others Washington, DC: American Society for Microbiology;
    [Google Scholar]
  38. Yarnell W.S., Roberts J.W. 1992; The phage ? gene Q transcription antiterminator binds DNA in the late gene promoter as it modifies RNA polymerase.. Cell 69:1181–1189
    [Google Scholar]
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