Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1994 Apr 11;22(7):1186–1193. doi: 10.1093/nar/22.7.1186

The use of a tRNA as a transcriptional reporter: the T7 late promoter is extremely efficient in Escherichia coli but its transcripts are poorly expressed.

P J Lopez 1, I Iost 1, M Dreyfus 1
PMCID: PMC523641  PMID: 8165132

Abstract

In gene expression studies, promoters are often fused to a protein-encoding reporter gene, the expression of which is then taken as an indirect measure of their strength. Here, we advocate the use of a tRNA reporter for the direct quantification of promoter strength. Using this method, we have studied the bacteriophage T7 gene 10 promoter in an E. coli strain that produces saturating amounts of T7 RNA polymerase. At 37 degrees C in aminoacid-glycerol medium, we show that this promoter ranks amongst the strongest known, directing ca 1.1 transcription events per second, 2.2-fold more than the promoters for rRNA operons, or 15-fold more than the induced lac promoter. Surprisingly, compared to the lac promoter, the T7 promoter is far less efficient in driving the expression of protein-encoding genes such as cat, neo or lacZ. Therefore, the polypeptide yield per transcript is lower when the T7 RNA polymerase is used instead of the E. coli RNA polymerase. The former enzyme travels faster than the translating ribosomes, and we suggest that this desynchronization lowers the polypeptide yield per transcript.

Full text

PDF
1186

Images in this article

1189Image
on p.1189
1190Image
on p.1190
1191Image
on p.1191
1191Image
on p.1191

Selected References

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

  1. Altman S., Kirsebom L., Talbot S. Recent studies of ribonuclease P. FASEB J. 1993 Jan;7(1):7–14. doi: 10.1096/fasebj.7.1.7916700. [DOI] [PubMed] [Google Scholar]
  2. Chamberlin M., Ring J. Characterization of T7-specific ribonucleic acid polymerase. 1. General properties of the enzymatic reaction and the template specificity of the enzyme. J Biol Chem. 1973 Mar 25;248(6):2235–2244. [PubMed] [Google Scholar]
  3. Chevrier-Miller M., Jacques N., Raibaud O., Dreyfus M. Transcription of single-copy hybrid lacZ genes by T7 RNA polymerase in Escherichia coli: mRNA synthesis and degradation can be uncoupled from translation. Nucleic Acids Res. 1990 Oct 11;18(19):5787–5792. doi: 10.1093/nar/18.19.5787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Christie G. E., Farnham P. J., Platt T. Synthetic sites for transcription termination and a functional comparison with tryptophan operon termination sites in vitro. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4180–4184. doi: 10.1073/pnas.78.7.4180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Crothers D. M., Cole P. E., Hilbers C. W., Shulman R. G. The molecular mechanism of thermal unfolding of Escherichia coli formylmethionine transfer RNA. J Mol Biol. 1974 Jul 25;87(1):63–88. doi: 10.1016/0022-2836(74)90560-9. [DOI] [PubMed] [Google Scholar]
  6. Davanloo P., Rosenberg A. H., Dunn J. J., Studier F. W. Cloning and expression of the gene for bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2035–2039. doi: 10.1073/pnas.81.7.2035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. DeFranco C., Schottel J. L. Terminal sequences do not contain the rate-limiting decay determinants of E. coli cat mRNA. Nucleic Acids Res. 1989 Feb 11;17(3):1139–1157. doi: 10.1093/nar/17.3.1139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Deuschle U., Kammerer W., Gentz R., Bujard H. Promoters of Escherichia coli: a hierarchy of in vivo strength indicates alternate structures. EMBO J. 1986 Nov;5(11):2987–2994. doi: 10.1002/j.1460-2075.1986.tb04596.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dreyfus M. What constitutes the signal for the initiation of protein synthesis on Escherichia coli mRNAs? J Mol Biol. 1988 Nov 5;204(1):79–94. doi: 10.1016/0022-2836(88)90601-8. [DOI] [PubMed] [Google Scholar]
  10. Emilsson V., Kurland C. G. Growth rate dependence of transfer RNA abundance in Escherichia coli. EMBO J. 1990 Dec;9(13):4359–4366. doi: 10.1002/j.1460-2075.1990.tb07885.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Emilsson V., Näslund A. K., Kurland C. G. Growth-rate-dependent accumulation of twelve tRNA species in Escherichia coli. J Mol Biol. 1993 Mar 20;230(2):483–491. doi: 10.1006/jmbi.1993.1165. [DOI] [PubMed] [Google Scholar]
  12. Golomb M., Chamberlin M. Characterization of T7-specific ribonucleic acid polymerase. IV. Resolution of the major in vitro transcripts by gel electrophoresis. J Biol Chem. 1974 May 10;249(9):2858–2863. [PubMed] [Google Scholar]
  13. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Iost I., Guillerez J., Dreyfus M. Bacteriophage T7 RNA polymerase travels far ahead of ribosomes in vivo. J Bacteriol. 1992 Jan;174(2):619–622. doi: 10.1128/jb.174.2.619-622.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jacquet M., Kepes A. Initiation, elongation and inactivation of lac messenger RNA in Escherichia coli studied studied by measurement of its beta-galactosidase synthesizing capacity in vivo. J Mol Biol. 1971 Sep 28;60(3):453–472. doi: 10.1016/0022-2836(71)90181-1. [DOI] [PubMed] [Google Scholar]
  16. Jørgensen F., Kurland C. G. Processivity errors of gene expression in Escherichia coli. J Mol Biol. 1990 Oct 20;215(4):511–521. doi: 10.1016/S0022-2836(05)80164-0. [DOI] [PubMed] [Google Scholar]
  17. Komine Y., Adachi T., Inokuchi H., Ozeki H. Genomic organization and physical mapping of the transfer RNA genes in Escherichia coli K12. J Mol Biol. 1990 Apr 20;212(4):579–598. doi: 10.1016/0022-2836(90)90224-A. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Lewicki B. T., Margus T., Remme J., Nierhaus K. H. Coupling of rRNA transcription and ribosomal assembly in vivo. Formation of active ribosomal subunits in Escherichia coli requires transcription of rRNA genes by host RNA polymerase which cannot be replaced by bacteriophage T7 RNA polymerase. J Mol Biol. 1993 Jun 5;231(3):581–593. doi: 10.1006/jmbi.1993.1311. [DOI] [PubMed] [Google Scholar]
  20. Macdonald L. E., Zhou Y., McAllister W. T. Termination and slippage by bacteriophage T7 RNA polymerase. J Mol Biol. 1993 Aug 20;232(4):1030–1047. doi: 10.1006/jmbi.1993.1458. [DOI] [PubMed] [Google Scholar]
  21. Maloy S. R., Nunn W. D. Selection for loss of tetracycline resistance by Escherichia coli. J Bacteriol. 1981 Feb;145(2):1110–1111. doi: 10.1128/jb.145.2.1110-1111.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. McClain W. H., Foss K., Jenkins R. A., Schneider J. Nucleotides that determine Escherichia coli tRNA(Arg) and tRNA(Lys) acceptor identities revealed by analyses of mutant opal and amber suppressor tRNAs. Proc Natl Acad Sci U S A. 1990 Dec;87(23):9260–9264. doi: 10.1073/pnas.87.23.9260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Milligan J. F., Groebe D. R., Witherell G. W., Uhlenbeck O. C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 1987 Nov 11;15(21):8783–8798. doi: 10.1093/nar/15.21.8783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Morse D. E., Yanofsky C. Polarity and the degradation of mRNA. Nature. 1969 Oct 25;224(5217):329–331. doi: 10.1038/224329a0. [DOI] [PubMed] [Google Scholar]
  26. Neidhardt F. C., Bloch P. L., Smith D. F. Culture medium for enterobacteria. J Bacteriol. 1974 Sep;119(3):736–747. doi: 10.1128/jb.119.3.736-747.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Petersen C. Control of functional mRNA stability in bacteria: multiple mechanisms of nucleolytic and non-nucleolytic inactivation. Mol Microbiol. 1992 Feb;6(3):277–282. doi: 10.1111/j.1365-2958.1992.tb01469.x. [DOI] [PubMed] [Google Scholar]
  28. Platt T. Transcription termination and the regulation of gene expression. Annu Rev Biochem. 1986;55:339–372. doi: 10.1146/annurev.bi.55.070186.002011. [DOI] [PubMed] [Google Scholar]
  29. Raibaud O., Mock M., Schwartz M. A technique for integrating any DNA fragment into the chromosome of Escherichia coli. Gene. 1984 Jul-Aug;29(1-2):231–241. doi: 10.1016/0378-1119(84)90183-5. [DOI] [PubMed] [Google Scholar]
  30. Reuven N. B., Deutscher M. P. Multiple exoribonucleases are required for the 3' processing of Escherichia coli tRNA precursors in vivo. FASEB J. 1993 Jan;7(1):143–148. doi: 10.1096/fasebj.7.1.8422961. [DOI] [PubMed] [Google Scholar]
  31. Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56(1):125–135. doi: 10.1016/0378-1119(87)90165-x. [DOI] [PubMed] [Google Scholar]
  32. Sampson J. R., Uhlenbeck O. C. Biochemical and physical characterization of an unmodified yeast phenylalanine transfer RNA transcribed in vitro. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1033–1037. doi: 10.1073/pnas.85.4.1033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Silhavy T. J., Beckwith J. R. Uses of lac fusions for the study of biological problems. Microbiol Rev. 1985 Dec;49(4):398–418. doi: 10.1128/mr.49.4.398-418.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Smith J. D. Transcription and processing of transfer RNA precursors. Prog Nucleic Acid Res Mol Biol. 1976;16:25–73. doi: 10.1016/s0079-6603(08)60755-2. [DOI] [PubMed] [Google Scholar]
  36. Studier F. W. Bacteriophage T7. Science. 1972 Apr 28;176(4033):367–376. doi: 10.1126/science.176.4033.367. [DOI] [PubMed] [Google Scholar]
  37. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  38. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Yarchuk O., Jacques N., Guillerez J., Dreyfus M. Interdependence of translation, transcription and mRNA degradation in the lacZ gene. J Mol Biol. 1992 Aug 5;226(3):581–596. doi: 10.1016/0022-2836(92)90617-s. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES