Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1987 Apr;169(4):1691–1701. doi: 10.1128/jb.169.4.1691-1701.1987

In vivo translation of a region within the rrnB 16S rRNA gene of Escherichia coli.

K L Berg, C L Squires, C Squires
PMCID: PMC212001  PMID: 2435709

Abstract

In this study we show that a segment of the Escherichia coli rrnB 16S gene can be translated in vivo. Other laboratories have previously reported that there are internal transcription and translation signals and open reading frames within the E. coli rrnB rRNA operon. Their studies revealed a translation start signal followed by a 252-base-pair open reading frame (ORF16) within the 16S gene and detected a promoter (p16) in the same general region by using in vitro RNA polymerase binding and transcription initiation assays. By using plasmid gene fusions of ORF16 to lacZ we showed that an ORF16'-'beta-galactosidase fusion protein was made in vivo. Transcripts encoding the fusion protein were expressed either from the rrnB p1p2 control region or from a hybrid trp-lac promoter (tacP), but the amount of expression was considerably less than for a lacZ control plasmid. We used fusions to the cat gene to show that p16 is one-half as active as lacP. Deletions were used to show that p16 is located within ORF16 and thus cannot promote a transcript encoding the ORF16 peptide. A comparison of sequences from different organisms shows that ORF16 and p16 lie in a highly conserved region of the procaryotic 16S RNA structure. The first 20 amino acids of ORF16 are conserved in most eubacterial and plant organellar sequences, and promoter activity has been detected in this region of the Caulobacter crescentus sequence by other workers.

Full text

PDF
1696

Images in this article

1695Image
on p.1695
1695Image
on p.1695

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aksoy S., Squires C. L., Squires C. Evidence for antitermination in Escherichia coli RRNA transcription. J Bacteriol. 1984 Jul;159(1):260–264. doi: 10.1128/jb.159.1.260-264.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amann E., Brosius J., Ptashne M. Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. Gene. 1983 Nov;25(2-3):167–178. doi: 10.1016/0378-1119(83)90222-6. [DOI] [PubMed] [Google Scholar]
  3. Amemiya K., Bellofatto V., Shapiro L., Feingold J. Transcription initiation in vitro and in vivo at a highly conserved promoter within a 16 S ribosomal RNA gene. J Mol Biol. 1986 Jan 5;187(1):1–14. doi: 10.1016/0022-2836(86)90401-8. [DOI] [PubMed] [Google Scholar]
  4. Berman M. L., Jackson D. E. Selection of lac gene fusions in vivo: ompR-lacZ fusions that define a functional domain of the ompR gene product. J Bacteriol. 1984 Aug;159(2):750–756. doi: 10.1128/jb.159.2.750-756.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boros I., Kiss A., Venetianer P. Physical map of the seven ribosomal RNA genes of Escherichia coli. Nucleic Acids Res. 1979;6(5):1817–1830. doi: 10.1093/nar/6.5.1817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brosius J., Dull T. J., Sleeter D. D., Noller H. F. Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. J Mol Biol. 1981 May 15;148(2):107–127. doi: 10.1016/0022-2836(81)90508-8. [DOI] [PubMed] [Google Scholar]
  7. Brosius J., Palmer M. L., Kennedy P. J., Noller H. F. Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4801–4805. doi: 10.1073/pnas.75.10.4801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brosius J. Plasmid vectors for the selection of promoters. Gene. 1984 Feb;27(2):151–160. doi: 10.1016/0378-1119(84)90136-7. [DOI] [PubMed] [Google Scholar]
  9. Carbon P., Ehresmann C., Ehresmann B., Ebel J. P. The complete nucleotide sequence of the ribosomal 16-S RNA from Excherichia coli. Experimental details and cistron heterogeneities. Eur J Biochem. 1979 Oct 15;100(2):399–410. doi: 10.1111/j.1432-1033.1979.tb04183.x. [DOI] [PubMed] [Google Scholar]
  10. Casadaban M. J., Chou J., Cohen S. N. In vitro gene fusions that join an enzymatically active beta-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J Bacteriol. 1980 Aug;143(2):971–980. doi: 10.1128/jb.143.2.971-980.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Feingold J., Bellofatto V., Shapiro L., Amemiya K. Organization and nucleotide sequence analysis of an rRNA and tRNA gene cluster from Caulobacter crescentus. J Bacteriol. 1985 Jul;163(1):155–166. doi: 10.1128/jb.163.1.155-166.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fiil N. P., Bendiak D., Collins J., Friesen J. D. Expression of Escherichia coli ribosomal protein and RNA polymerase genes cloned on plasmids. Mol Gen Genet. 1979 May 23;173(1):39–50. doi: 10.1007/BF00267689. [DOI] [PubMed] [Google Scholar]
  13. Fuller F. A family of cloning vectors containing the lacUV5 promoter. Gene. 1982 Jul-Aug;19(1):43–54. doi: 10.1016/0378-1119(82)90187-1. [DOI] [PubMed] [Google Scholar]
  14. Holben W. E., Prasad S. M., Morgan E. A. Antitermination by both the promoter and the leader regions of an Escherichia coli ribosomal RNA operon. Proc Natl Acad Sci U S A. 1985 Aug;82(15):5073–5077. doi: 10.1073/pnas.82.15.5073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Howard P. K., Shaw J., Otsuka A. J. Nucleotide sequence of the birA gene encoding the biotin operon repressor and biotin holoenzyme synthetase functions of Escherichia coli. Gene. 1985;35(3):321–331. doi: 10.1016/0378-1119(85)90011-3. [DOI] [PubMed] [Google Scholar]
  16. Huysmans E., De Wachter R. Compilation of small ribosomal subunit RNA sequences. Nucleic Acids Res. 1986;14 (Suppl):r73–118. doi: 10.1093/nar/14.suppl.r73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kirschbaum J. B., Konrad E. B. Isolation of a specialized lambda transducing bacteriophage carrying the beta subunit gene for Escherichia coli ribonucleic acid polymerase. J Bacteriol. 1973 Nov;116(2):517–526. doi: 10.1128/jb.116.2.517-526.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Konigsberg W., Godson G. N. Evidence for use of rare codons in the dnaG gene and other regulatory genes of Escherichia coli. Proc Natl Acad Sci U S A. 1983 Feb;80(3):687–691. doi: 10.1073/pnas.80.3.687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kop J., Kopylov A. M., Magrum L., Siegel R., Gupta R., Woese C. R., Noller H. F. Probing the structure of 16 S ribosomal RNA from Bacillus brevis. J Biol Chem. 1984 Dec 25;259(24):15287–15293. [PubMed] [Google Scholar]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. Li S. C., Squires C. L., Squires C. Antitermination of E. coli rRNA transcription is caused by a control region segment containing lambda nut-like sequences. Cell. 1984 Oct;38(3):851–860. doi: 10.1016/0092-8674(84)90280-0. [DOI] [PubMed] [Google Scholar]
  22. Lipman D. J., Pearson W. R. Rapid and sensitive protein similarity searches. Science. 1985 Mar 22;227(4693):1435–1441. doi: 10.1126/science.2983426. [DOI] [PubMed] [Google Scholar]
  23. Marinus M. G. Location of DNA methylation genes on the Escherichia coli K-12 genetic map. Mol Gen Genet. 1973 Dec 14;127(1):47–55. doi: 10.1007/BF00267782. [DOI] [PubMed] [Google Scholar]
  24. Messing J., Gronenborn B., Müller-Hill B., Hans Hopschneider P. Filamentous coliphage M13 as a cloning vehicle: insertion of a HindII fragment of the lac regulatory region in M13 replicative form in vitro. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3642–3646. doi: 10.1073/pnas.74.9.3642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mulligan M. E., Hawley D. K., Entriken R., McClure W. R. Escherichia coli promoter sequences predict in vitro RNA polymerase selectivity. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 2):789–800. doi: 10.1093/nar/12.1part2.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Munson L. M., Stormo G. D., Niece R. L., Reznikoff W. S. lacZ translation initiation mutations. J Mol Biol. 1984 Aug 25;177(4):663–683. doi: 10.1016/0022-2836(84)90043-3. [DOI] [PubMed] [Google Scholar]
  27. Nilsson G., Belasco J. G., Cohen S. N., von Gabain A. Growth-rate dependent regulation of mRNA stability in Escherichia coli. Nature. 1984 Nov 1;312(5989):75–77. doi: 10.1038/312075a0. [DOI] [PubMed] [Google Scholar]
  28. Nomura M., Gourse R., Baughman G. Regulation of the synthesis of ribosomes and ribosomal components. Annu Rev Biochem. 1984;53:75–117. doi: 10.1146/annurev.bi.53.070184.000451. [DOI] [PubMed] [Google Scholar]
  29. Sancar A., Hack A. M., Rupp W. D. Simple method for identification of plasmid-coded proteins. J Bacteriol. 1979 Jan;137(1):692–693. doi: 10.1128/jb.137.1.692-693.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sarmientos P., Sylvester J. E., Contente S., Cashel M. Differential stringent control of the tandem E. coli ribosomal RNA promoters from the rrnA operon expressed in vivo in multicopy plasmids. Cell. 1983 Apr;32(4):1337–1346. doi: 10.1016/0092-8674(83)90314-8. [DOI] [PubMed] [Google Scholar]
  31. Sekiya T., Nishimura S. Sequence of the gene for isoleucine tRNA1 and the surrounding region in a ribosomal RNA operon of Escherichia coli. Nucleic Acids Res. 1979 Feb;6(2):575–592. doi: 10.1093/nar/6.2.575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Shen W. F., Squires C., Squires C. L. Nucleotide sequence of the rrnG ribosomal RNA promoter region of Escherichia coli. Nucleic Acids Res. 1982 May 25;10(10):3303–3313. doi: 10.1093/nar/10.10.3303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. doi: 10.1073/pnas.71.4.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Squires C., Krainer A., Barry G., Shen W. F., Squires C. L. Nucleotide sequence at the end of the gene for the RNA polymerase beta' subunit (rpoC). Nucleic Acids Res. 1981 Dec 21;9(24):6827–6840. doi: 10.1093/nar/9.24.6827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Stüber D., Bujard H. Organization of transcriptional signals in plasmids pBR322 and pACYC184. Proc Natl Acad Sci U S A. 1981 Jan;78(1):167–171. doi: 10.1073/pnas.78.1.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Taylor W. E., Burgess R. R. Escherichia coli RNA polymerase binding and initiation of transcription on fragments of lambda rifd 18 DNA containing promoters for lambda genes and for rrnB, tufB, rplC,A, rplJ,L, and rpoB,C genes. Gene. 1979 Aug;6(4):331–365. doi: 10.1016/0378-1119(79)90073-8. [DOI] [PubMed] [Google Scholar]
  37. Woese C. R., Gutell R., Gupta R., Noller H. F. Detailed analysis of the higher-order structure of 16S-like ribosomal ribonucleic acids. Microbiol Rev. 1983 Dec;47(4):621–669. doi: 10.1128/mr.47.4.621-669.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Young R. A., Macklis R., Steitz J. A. Sequence of the 16 S-23 s spacer region in two ribosomal RNA operons of Escherichia coli. J Biol Chem. 1979 May 10;254(9):3264–3271. [PubMed] [Google Scholar]
  39. Young R. A., Steitz J. A. Complementary sequences 1700 nucleotides apart form a ribonuclease III cleavage site in Escherichia coli ribosomal precursor RNA. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3593–3597. doi: 10.1073/pnas.75.8.3593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Young R. A., Steitz J. A. Tandem promoters direct E. coli ribosomal RNA synthesis. Cell. 1979 May;17(1):225–234. doi: 10.1016/0092-8674(79)90310-6. [DOI] [PubMed] [Google Scholar]
  41. de Boer H. A., Gilbert S. F., Nomura M. DNA sequences of promoter regions for rRNA operons rrnE and rrnA in E. coli. Cell. 1979 May;17(1):201–209. doi: 10.1016/0092-8674(79)90308-8. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES