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. 1986 Sep;83(17):6548–6552. doi: 10.1073/pnas.83.17.6548

Construction of two Escherichia coli amber suppressor genes: tRNAPheCUA and tRNACysCUA.

J Normanly, J M Masson, L G Kleina, J Abelson, J H Miller
PMCID: PMC386541  PMID: 3529087

Abstract

Amber suppressor genes corresponding to Escherichia coli tRNAPhe and tRNACys have been constructed for use in amino acid substitution studies as well as protein engineering. The genes for either tRNAPheGAA or tRNACysGCA both with the anticodon 5' CTA 3' were assembled from four to six oligonucleotides, which were annealed and ligated into a vector. The suppressor genes are expressed constitutively from a synthetic promoter, derived from the promoter sequence of the E. coli lipoprotein gene. The tRNAPhe suppressor (tRNAPheCUA) is 54-100% efficient in vivo, while the tRNACys suppressor (tRNACysCUA) is 17-50% efficient. To verify that the suppressors insert the predicted amino acids, both genes were used to suppress an amber mutation in a protein coding sequence. NH2-terminal sequence analysis of the resultant proteins revealed that tRNAPheCUA and tRNACysCUA insert phenylalanine and cysteine, respectively. To demonstrate the potential of these suppressors, tRNAPheCUA and tRNACysCUA have been used to effect amino acid substitutions at specific sites in the E. coli lac repressor.

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Selected References

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

  1. Abelson J. RNA processing and the intervening sequence problem. Annu Rev Biochem. 1979;48:1035–1069. doi: 10.1146/annurev.bi.48.070179.005131. [DOI] [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. Baccanari D. P., Averett D., Briggs C., Burchall J. Escherichia coli dihydrofolate reductase: isolation and characterization of two isozymes. Biochemistry. 1977 Aug 9;16(16):3566–3572. doi: 10.1021/bi00635a010. [DOI] [PubMed] [Google Scholar]
  4. Baccanari D. P., Stone D., Kuyper L. Effect of a single amino acid substitution on Escherichia coli dihydrofolate reductase catalysis and ligand binding. J Biol Chem. 1981 Feb 25;256(4):1738–1747. [PubMed] [Google Scholar]
  5. Baccanari D., Phillips A., Smith S., Sinski D., Burchall J. Purification and properties of Escherichia coli dihydrofolate reductase. Biochemistry. 1975 Dec 2;14(24):5267–5273. doi: 10.1021/bi00695a006. [DOI] [PubMed] [Google Scholar]
  6. Barrell B. G., Sanger F. The sequence of phenylalanine tRNA from E. coli. FEBS Lett. 1969 Jun;3(4):275–278. doi: 10.1016/0014-5793(69)80157-2. [DOI] [PubMed] [Google Scholar]
  7. Bolin J. T., Filman D. J., Matthews D. A., Hamlin R. C., Kraut J. Crystal structures of Escherichia coli and Lactobacillus casei dihydrofolate reductase refined at 1.7 A resolution. I. General features and binding of methotrexate. J Biol Chem. 1982 Nov 25;257(22):13650–13662. [PubMed] [Google Scholar]
  8. Bossi L. Context effects: translation of UAG codon by suppressor tRNA is affected by the sequence following UAG in the message. J Mol Biol. 1983 Feb 15;164(1):73–87. doi: 10.1016/0022-2836(83)90088-8. [DOI] [PubMed] [Google Scholar]
  9. Capone J. P., Sharp P. A., RajBhandary U. L. Amber, ochre and opal suppressor tRNA genes derived from a human serine tRNA gene. EMBO J. 1985 Jan;4(1):213–221. doi: 10.1002/j.1460-2075.1985.tb02338.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dente L., Cesareni G., Cortese R. pEMBL: a new family of single stranded plasmids. Nucleic Acids Res. 1983 Mar 25;11(6):1645–1655. doi: 10.1093/nar/11.6.1645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Deutscher M. P. Processing of tRNA in prokaryotes and eukaryotes. CRC Crit Rev Biochem. 1984;17(1):45–71. doi: 10.3109/10409238409110269. [DOI] [PubMed] [Google Scholar]
  12. Filman D. J., Bolin J. T., Matthews D. A., Kraut J. Crystal structures of Escherichia coli and Lactobacillus casei dihydrofolate reductase refined at 1.7 A resolution. II. Environment of bound NADPH and implications for catalysis. J Biol Chem. 1982 Nov 25;257(22):13663–13672. [PubMed] [Google Scholar]
  13. Fowler A. V., Zabin I. Purification, structure, and properties of hybrid beta-galactosidase proteins. J Biol Chem. 1983 Dec 10;258(23):14354–14358. [PubMed] [Google Scholar]
  14. Goodman H. M., Abelson J., Landy A., Brenner S., Smith J. D. Amber suppression: a nucleotide change in the anticodon of a tyrosine transfer RNA. Nature. 1968 Mar 16;217(5133):1019–1024. doi: 10.1038/2171019a0. [DOI] [PubMed] [Google Scholar]
  15. Gorini L. Informational suppression. Annu Rev Genet. 1970;4:107–134. doi: 10.1146/annurev.ge.04.120170.000543. [DOI] [PubMed] [Google Scholar]
  16. Hewick R. M., Hunkapiller M. W., Hood L. E., Dreyer W. J. A gas-liquid solid phase peptide and protein sequenator. J Biol Chem. 1981 Aug 10;256(15):7990–7997. [PubMed] [Google Scholar]
  17. Hudziak R. M., Laski F. A., RajBhandary U. L., Sharp P. A., Capecchi M. R. Establishment of mammalian cell lines containing multiple nonsense mutations and functional suppressor tRNA genes. Cell. 1982 Nov;31(1):137–146. doi: 10.1016/0092-8674(82)90413-5. [DOI] [PubMed] [Google Scholar]
  18. Inokuchi H., Yamao F., Sakano H., Ozeki H. Identification of transfer RNA suppressors in Escherichia coli. I. Amber suppressor su+2, an anticodon mutant of tRNA2Gln. J Mol Biol. 1979 Aug 25;132(4):649–662. doi: 10.1016/0022-2836(79)90380-2. [DOI] [PubMed] [Google Scholar]
  19. Khorana H. G. Total synthesis of a gene. Science. 1979 Feb 16;203(4381):614–625. doi: 10.1126/science.366749. [DOI] [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. Laski F. A., Belagaje R., Hudziak R. M., Capecchi M. R., Norton G. P., Palese P., RajBhandary U. L., Sharp P. A. Synthesis of an ochre suppressor tRNA gene and expression in mammalian cells. EMBO J. 1984 Nov;3(11):2445–2452. doi: 10.1002/j.1460-2075.1984.tb02154.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Laski F. A., Belagaje R., RajBhandary U. L., Sharp P. A. An amber suppressor tRNA gene derived by site-specific mutagenesis: cloning and function in mammalian cells. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5813–5817. doi: 10.1073/pnas.79.19.5813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Matthews D. A., Alden R. A., Bolin J. T., Freer S. T., Hamlin R., Xuong N., Kraut J., Poe M., Williams M., Hoogsteen K. Dihydrofolate reductase: x-ray structure of the binary complex with methotrexate. Science. 1977 Jul 29;197(4302):452–455. doi: 10.1126/science.17920. [DOI] [PubMed] [Google Scholar]
  24. Mazzara G. P., McClain W. H. Cysteine transfer RNA of Escherichia coli: nucleotide sequence and unusual metabolic properties of the 3' C-C-A terminus. J Mol Biol. 1977 Dec 25;117(4):1061–1079. doi: 10.1016/s0022-2836(77)80013-2. [DOI] [PubMed] [Google Scholar]
  25. Miller J. H., Albertini A. M. Effects of surrounding sequence on the suppression of nonsense codons. J Mol Biol. 1983 Feb 15;164(1):59–71. doi: 10.1016/0022-2836(83)90087-6. [DOI] [PubMed] [Google Scholar]
  26. Miller J. H., Coulondre C., Hofer M., Schmeissner U., Sommer H., Schmitz A., Lu P. Genetic studies of the lac repressor. IX. Generation of altered proteins by the suppression of nonsence mutations. J Mol Biol. 1979 Jun 25;131(2):191–222. doi: 10.1016/0022-2836(79)90073-1. [DOI] [PubMed] [Google Scholar]
  27. Murgola E. J., Prather N. E., Pagel F. T., Mims B. H., Hijazi K. A. Missense and nonsense suppressors derived from a glycine tRNA by nucleotide insertion and deletion in vivo. Mol Gen Genet. 1984;193(1):76–81. doi: 10.1007/BF00327417. [DOI] [PubMed] [Google Scholar]
  28. Nakamura K., Inouye M. DNA sequence of the gene for the outer membrane lipoprotein of E. coli: an extremely AT-rich promoter. Cell. 1979 Dec;18(4):1109–1117. doi: 10.1016/0092-8674(79)90224-1. [DOI] [PubMed] [Google Scholar]
  29. Newman A. J., Ogden R. C., Abelson J. tRNA gene transcription in yeast: effects of specified base substitutions in the intragenic promoter. Cell. 1983 Nov;35(1):117–125. doi: 10.1016/0092-8674(83)90214-3. [DOI] [PubMed] [Google Scholar]
  30. Normanly J., Ogden R. C., Horvath S. J., Abelson J. Changing the identity of a transfer RNA. Nature. 1986 May 15;321(6067):213–219. doi: 10.1038/321213a0. [DOI] [PubMed] [Google Scholar]
  31. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Schulman L. H., Pelka H. Anticodon loop size and sequence requirements for recognition of formylmethionine tRNA by methionyl-tRNA synthetase. Proc Natl Acad Sci U S A. 1983 Nov;80(22):6755–6759. doi: 10.1073/pnas.80.22.6755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schulman L. H., Pelka H., Susani M. Base substitutions in the wobble position of the anticodon inhibit aminoacylation of E. coli tRNAfMet by E. coli Met-tRNA synthetase. Nucleic Acids Res. 1983 Mar 11;11(5):1439–1455. doi: 10.1093/nar/11.5.1439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schulman L. H. Structure and function of Escherichia coli formylmethionine transfer RNA: loss of methionine acceptor activity by modification of a specific guanosine residue in the acceptor stem of formylmethionine transfer RNA from Escherichia coli. Proc Natl Acad Sci U S A. 1972 Dec;69(12):3594–3597. doi: 10.1073/pnas.69.12.3594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Smith D. R., Calvo J. M. Nucleotide sequence of the E coli gene coding for dihydrofolate reductase. Nucleic Acids Res. 1980 May 24;8(10):2255–2274. doi: 10.1093/nar/8.10.2255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Steege D. A. A nucleotide change in the anticodon of an Escherichia coli serine transfer RNA results in supD-amber suppression. Nucleic Acids Res. 1983 Jun 11;11(11):3823–3832. doi: 10.1093/nar/11.11.3823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Steitz T. A., Weber I. T., Matthew J. B. Catabolite gene activator protein: structure, homology with other proteins, and cyclic AMP and DNA binding. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 1):419–426. doi: 10.1101/sqb.1983.047.01.049. [DOI] [PubMed] [Google Scholar]
  38. Thorbjarnardóttir S., Dingermann T., Rafnar T., Andrésson O. S., Söll D., Eggertsson G. Leucine tRNA family of Escherichia coli: nucleotide sequence of the supP(Am) suppressor gene. J Bacteriol. 1985 Jan;161(1):219–222. doi: 10.1128/jb.161.1.219-222.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Villafranca J. E., Howell E. E., Voet D. H., Strobel M. S., Ogden R. C., Abelson J. N., Kraut J. Directed mutagenesis of dihydrofolate reductase. Science. 1983 Nov 18;222(4625):782–788. doi: 10.1126/science.6356360. [DOI] [PubMed] [Google Scholar]
  40. Wray W., Boulikas T., Wray V. P., Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem. 1981 Nov 15;118(1):197–203. doi: 10.1016/0003-2697(81)90179-2. [DOI] [PubMed] [Google Scholar]
  41. Yaniv M., Folk W. R., Berg P., Soll L. A single mutational modification of a tryptophan-specific transfer RNA permits aminoacylation by glutamine and translation of the codon UAG. J Mol Biol. 1974 Jun 25;86(2):245–260. doi: 10.1016/0022-2836(74)90016-3. [DOI] [PubMed] [Google Scholar]
  42. Yoshimura M., Inokuchi H., Ozeki H. Identification of transfer RNA suppressors in Escherichia coli. IV. Amber suppressor Su+6 a double mutant of a new species of leucine tRNA. J Mol Biol. 1984 Aug 25;177(4):627–644. doi: 10.1016/0022-2836(84)90041-x. [DOI] [PubMed] [Google Scholar]
  43. Young R. A. Transcription termination in the Escherichia coli ribosomal RNA operon rrnC. J Biol Chem. 1979 Dec 25;254(24):12725–12731. [PubMed] [Google Scholar]

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