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. 1991 Sep 11;19(17):4595–4600. doi: 10.1093/nar/19.17.4595

Lack of a 5' non-coding region in Tn1721 encoded tetR mRNA is associated with a low efficiency of translation and a short half-life in Escherichia coli.

R Baumeister 1, P Flache 1, O Melefors 1, A von Gabain 1, W Hillen 1
PMCID: PMC328697  PMID: 1653948

Abstract

The repressor-encoding tetR gene from Tn1721 is expressed with a very low efficiency. Its mRNA lacks an untranslated leader sequence. We have constructed protein fusions with the lacZ gene which contain between 14 and 157 5' nucleotides from the tetR gene. Since they are all expressed with similar efficiencies we conclude that the sequence information for initiation of translation is contained within the first 14 bases of the tetR coding region. These fusion transcripts are about 20-fold less efficiently translated than the wild type lacZ transcript. A toeprint analysis confirms that the initiation complex is indistinguishable from those formed by regular transcripts with 5' untranslated regions but occurs in a very low amount in vitro. Thus, the absence of a 5' leader causes a poor rate of translation initiation. The half-lives of tetR and tetR-lacZ mRNAs are about 30 seconds, which is 3-times lower than that of the wt lacZ mRNA. Inactivation of the ams/rne locus in E. coli stabilizes the tetR transcript more than ten-fold. The influence of translation on the tetR half-life is discussed.

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Selected References

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

  1. Altenbuchner J., Schmid K., Schmitt R. Tn1721-encoded tetracycline resistance: mapping of structural and regulatory genes mediating resistance. J Bacteriol. 1983 Jan;153(1):116–123. doi: 10.1128/jb.153.1.116-123.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Altschmied L., Baumeister R., Pfleiderer K., Hillen W. A threonine to alanine exchange at position 40 of Tet repressor alters the recognition of the sixth base pair of tet operator from GC to AT. EMBO J. 1988 Dec 1;7(12):4011–4017. doi: 10.1002/j.1460-2075.1988.tb03290.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ayer D. E., Dynan W. S. Simian virus 40 major late promoter: a novel tripartite structure that includes intragenic sequences. Mol Cell Biol. 1988 May;8(5):2021–2033. doi: 10.1128/mcb.8.5.2021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Babitzke P., Kushner S. R. The Ams (altered mRNA stability) protein and ribonuclease E are encoded by the same structural gene of Escherichia coli. Proc Natl Acad Sci U S A. 1991 Jan 1;88(1):1–5. doi: 10.1073/pnas.88.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Belasco J. G., Higgins C. F. Mechanisms of mRNA decay in bacteria: a perspective. Gene. 1988 Dec 10;72(1-2):15–23. doi: 10.1016/0378-1119(88)90123-0. [DOI] [PubMed] [Google Scholar]
  6. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  7. Calogero R. A., Pon C. L., Canonaco M. A., Gualerzi C. O. Selection of the mRNA translation initiation region by Escherichia coli ribosomes. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6427–6431. doi: 10.1073/pnas.85.17.6427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cannistraro V. J., Subbarao M. N., Kennell D. Specific endonucleolytic cleavage sites for decay of Escherichia coli mRNA. J Mol Biol. 1986 Nov 20;192(2):257–274. doi: 10.1016/0022-2836(86)90363-3. [DOI] [PubMed] [Google Scholar]
  9. Chauhan A. K., Miczak A., Taraseviciene L., Apirion D. Sequencing and expression of the rne gene of Escherichia coli. Nucleic Acids Res. 1991 Jan 11;19(1):125–129. doi: 10.1093/nar/19.1.125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Claverie-Martin F., Diaz-Torres M. R., Yancey S. D., Kushner S. R. Analysis of the altered mRNA stability (ams) gene from Escherichia coli. Nucleotide sequence, transcriptional analysis, and homology of its product to MRP3, a mitochondrial ribosomal protein from Neurospora crassa. J Biol Chem. 1991 Feb 15;266(5):2843–2851. [PubMed] [Google Scholar]
  11. Gheysen D., Iserentant D., Derom C., Fiers W. Systematic alteration of the nucleotide sequence preceding the translation initiation codon and the effects on bacterial expression of the cloned SV40 small-t antigen gene. Gene. 1982 Jan;17(1):55–63. doi: 10.1016/0378-1119(82)90100-7. [DOI] [PubMed] [Google Scholar]
  12. Gold L. Posttranscriptional regulatory mechanisms in Escherichia coli. Annu Rev Biochem. 1988;57:199–233. doi: 10.1146/annurev.bi.57.070188.001215. [DOI] [PubMed] [Google Scholar]
  13. Goldblum K., Apririon D. Inactivation of the ribonucleic acid-processing enzyme ribonuclease E blocks cell division. J Bacteriol. 1981 Apr;146(1):128–132. doi: 10.1128/jb.146.1.128-132.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gualerzi C. O., Pon C. L. Initiation of mRNA translation in prokaryotes. Biochemistry. 1990 Jun 26;29(25):5881–5889. doi: 10.1021/bi00477a001. [DOI] [PubMed] [Google Scholar]
  15. Hartz D., Binkley J., Hollingsworth T., Gold L. Domains of initiator tRNA and initiation codon crucial for initiator tRNA selection by Escherichia coli IF3. Genes Dev. 1990 Oct;4(10):1790–1800. doi: 10.1101/gad.4.10.1790. [DOI] [PubMed] [Google Scholar]
  16. Hartz D., McPheeters D. S., Traut R., Gold L. Extension inhibition analysis of translation initiation complexes. Methods Enzymol. 1988;164:419–425. doi: 10.1016/s0076-6879(88)64058-4. [DOI] [PubMed] [Google Scholar]
  17. Ippen K., Shapiro J. A., Beckwith J. R. Transposition of the lac region to the gal region of the Escherichia coli chromosome: isolation of lambda-lac transducing bacteriophages. J Bacteriol. 1971 Oct;108(1):5–9. doi: 10.1128/jb.108.1.5-9.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jacques N., Dreyfus M. Translation initiation in Escherichia coli: old and new questions. Mol Microbiol. 1990 Jul;4(7):1063–1067. doi: 10.1111/j.1365-2958.1990.tb00679.x. [DOI] [PubMed] [Google Scholar]
  19. Klock G., Hillen W. Expression, purification and operator binding of the transposon Tn1721-encoded Tet repressor. J Mol Biol. 1986 Jun 20;189(4):633–641. doi: 10.1016/0022-2836(86)90493-6. [DOI] [PubMed] [Google Scholar]
  20. Lim L. W., Kennell D. Models for decay of Escherichia coli lac messenger RNA and evidence for inactivating cleavages between its messages. J Mol Biol. 1979 Dec 5;135(2):369–390. doi: 10.1016/0022-2836(79)90442-x. [DOI] [PubMed] [Google Scholar]
  21. Lundberg U., von Gabain A., Melefors O. Cleavages in the 5' region of the ompA and bla mRNA control stability: studies with an E. coli mutant altering mRNA stability and a novel endoribonuclease. EMBO J. 1990 Sep;9(9):2731–2741. doi: 10.1002/j.1460-2075.1990.tb07460.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Melançon P., Leclerc D., Brakier-Gingras L. A deletion mutation at the 5' end of Escherichia coli 16S ribosomal RNA. Biochim Biophys Acta. 1990 Aug 27;1050(1-3):98–103. doi: 10.1016/0167-4781(90)90148-u. [DOI] [PubMed] [Google Scholar]
  23. Melançon P., Leclerc D., Destroismaisons N., Brakier-Gingras L. The anti-Shine-Dalgarno region in Escherichia coli 16S ribosomal RNA is not essential for the correct selection of translational starts. Biochemistry. 1990 Apr 3;29(13):3402–3407. doi: 10.1021/bi00465a037. [DOI] [PubMed] [Google Scholar]
  24. Melefors O., von Gabain A. Genetic studies of cleavage-initiated mRNA decay and processing of ribosomal 9S RNA show that the Escherichia coli ams and rne loci are the same. Mol Microbiol. 1991 Apr;5(4):857–864. doi: 10.1111/j.1365-2958.1991.tb00759.x. [DOI] [PubMed] [Google Scholar]
  25. Melefors O., von Gabain A. Site-specific endonucleolytic cleavages and the regulation of stability of E. coli ompA mRNA. Cell. 1988 Mar 25;52(6):893–901. doi: 10.1016/0092-8674(88)90431-x. [DOI] [PubMed] [Google Scholar]
  26. Mudd E. A., Krisch H. M., Higgins C. F. RNase E, an endoribonuclease, has a general role in the chemical decay of Escherichia coli mRNA: evidence that rne and ams are the same genetic locus. Mol Microbiol. 1990 Dec;4(12):2127–2135. doi: 10.1111/j.1365-2958.1990.tb00574.x. [DOI] [PubMed] [Google Scholar]
  27. Mudd E. A., Prentki P., Belin D., Krisch H. M. Processing of unstable bacteriophage T4 gene 32 mRNAs into a stable species requires Escherichia coli ribonuclease E. EMBO J. 1988 Nov;7(11):3601–3607. doi: 10.1002/j.1460-2075.1988.tb03238.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Ono M., Kuwano M. A conditional lethal mutation in an Escherichia coli strain with a longer chemical lifetime of messenger RNA. J Mol Biol. 1979 Apr 15;129(3):343–357. doi: 10.1016/0022-2836(79)90500-x. [DOI] [PubMed] [Google Scholar]
  30. Petersen C. The functional stability of the lacZ transcript is sensitive towards sequence alterations immediately downstream of the ribosome binding site. Mol Gen Genet. 1987 Aug;209(1):179–187. doi: 10.1007/BF00329856. [DOI] [PubMed] [Google Scholar]
  31. Petersen G. B., Stockwell P. A., Hill D. F. Messenger RNA recognition in Escherichia coli: a possible second site of interaction with 16S ribosomal RNA. EMBO J. 1988 Dec 1;7(12):3957–3962. doi: 10.1002/j.1460-2075.1988.tb03282.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ptashne M., Backman K., Humayun M. Z., Jeffrey A., Maurer R., Meyer B., Sauer R. T. Autoregulation and function of a repressor in bacteriophage lambda. Science. 1976 Oct 8;194(4261):156–161. doi: 10.1126/science.959843. [DOI] [PubMed] [Google Scholar]
  33. Schauder B., McCarthy J. E. The role of bases upstream of the Shine-Dalgarno region and in the coding sequence in the control of gene expression in Escherichia coli: translation and stability of mRNAs in vivo. Gene. 1989 May 15;78(1):59–72. doi: 10.1016/0378-1119(89)90314-4. [DOI] [PubMed] [Google Scholar]
  34. Scherer G. F., Walkinshaw M. D., Arnott S., Morré D. J. The ribosome binding sites recognized by E. coli ribosomes have regions with signal character in both the leader and protein coding segments. Nucleic Acids Res. 1980 Sep 11;8(17):3895–3907. doi: 10.1093/nar/8.17.3895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schneider E., Blundell M., Kennell D. Translation and mRNA decay. Mol Gen Genet. 1978 Apr 6;160(2):121–129. doi: 10.1007/BF00267473. [DOI] [PubMed] [Google Scholar]
  36. Schneider T. D., Stormo G. D., Gold L., Ehrenfeucht A. Information content of binding sites on nucleotide sequences. J Mol Biol. 1986 Apr 5;188(3):415–431. doi: 10.1016/0022-2836(86)90165-8. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Sprengart M. L., Fatscher H. P., Fuchs E. The initiation of translation in E. coli: apparent base pairing between the 16srRNA and downstream sequences of the mRNA. Nucleic Acids Res. 1990 Apr 11;18(7):1719–1723. doi: 10.1093/nar/18.7.1719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Stormo G. D., Schneider T. D., Gold L. M. Characterization of translational initiation sites in E. coli. Nucleic Acids Res. 1982 May 11;10(9):2971–2996. doi: 10.1093/nar/10.9.2971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tai P. C., Wallace B. J., Davis B. D. Actions of aurintricarboxylate, kasugamycin, and pactamycin on Escherichia coli polysomes. Biochemistry. 1973 Feb;12(4):616–620. doi: 10.1021/bi00728a008. [DOI] [PubMed] [Google Scholar]
  41. Talkad V., Schneider E., Kennell D. Evidence for variable rates of ribosome movement in Escherichia coli. J Mol Biol. 1976 Jun 14;104(1):299–303. doi: 10.1016/0022-2836(76)90015-2. [DOI] [PubMed] [Google Scholar]
  42. Uzan M., Favre R., Brody E. A nuclease that cuts specifically in the ribosome binding site of some T4 mRNAs. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8895–8899. doi: 10.1073/pnas.85.23.8895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Walz A., Pirrotta V., Ineichen K. Lambda repressor regulates the switch between PR and Prm promoters. Nature. 1976 Aug 19;262(5570):665–669. doi: 10.1038/262665a0. [DOI] [PubMed] [Google Scholar]
  44. Wissmann A., Meier I., Wray L. V., Jr, Geissendörfer M., Hillen W. Tn10 tet operator mutations affecting Tet repressor recognition. Nucleic Acids Res. 1986 May 27;14(10):4253–4266. doi: 10.1093/nar/14.10.4253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. Yu X. M., Reznikoff W. S. Deletion analysis of the CAP-cAMP binding site of the Escherichia coli lactose promoter. Nucleic Acids Res. 1984 Jul 11;12(13):5449–5464. doi: 10.1093/nar/12.13.5449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. von Gabain A., Belasco J. G., Schottel J. L., Chang A. C., Cohen S. N. Decay of mRNA in Escherichia coli: investigation of the fate of specific segments of transcripts. Proc Natl Acad Sci U S A. 1983 Feb;80(3):653–657. doi: 10.1073/pnas.80.3.653. [DOI] [PMC free article] [PubMed] [Google Scholar]

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