Summary
Two mutants of Escherichia coli, trmC1 and trmC2, which are both defective in the synthesis of 5-methylaminomethyl-2-thiouridine (mnm5s2U) were utilized to study the function of this complex modified nucleoside. Transfer RNAs specific for glutamine, glutamic acid and lysine as well as a specific ochre suppressor derived from lysine tRNA (tRNA lysUAA encoded by the supG allele), contain this modified nucleoside at position 34 (the wobble position). It was found that two different undermodified derivatives of mnm5s2U were present in the two trmC mutants, which suggests that the two mutations affect two different enzymatic activities. Using the lacI-Z fusion system (Miller and Albertini 1983), we found that the efficiency of supG-mediated suppression was reduced to 30%–90% of the wild-type value in the trmC mutants. The modificationdeficient supG-tRNA in the mutants showed a higher sensitivity to codon context than the normal tRNA lysUAA .
Similar content being viewed by others
References
Agris PF, Söll D, Seno T (1973) Biological function of 2-thiouridine in Escherichia coli glutamic acid transfer ribonucleic acid. Biochemistry 12:4331–4337
Akaboshi E, Inouye M, Tsugita A (1976) Effect of neighboring nucleotide sequences on suppression efficiency in amber mutants of T4 phage lysozyme. Mol Gen Genet 149:1–4
Avital S, Elson D (1969) A convenient procedure for preparing transfer ribonucleic acid from Escherichia coli. Biochim Biophys Acta 179:297–307
Bare L, Bruce AG, Gesteland R, Uhlenbeck OC (1983) Uridine-33 in yeast tRNA not essential for amber suppression. Nature 305:554–556
Bertani G (1951) Studies of lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol 62:293–300
Björk GR, Kjellin-Stråby K (1978) Escherichia coli mutants with defects in the biosynthesis of 5-methylaminomethyl-2-thiouridine or 1-methylguanosine in their tRNA. J Bacteriol 133:508–517
Björk GR (1984) Modified nucleosides in RNA — their formation and function. In: Apirion D (ed) Processing of RNA. CRC Press, Boca Raton, pp 291–330
Bossi L (1983) Context effects: Translation of UAG codon by suppressor tRNA is affected by the sequence following UAG in the message. J Mol Biol 164:73–87
Bossi L, Roth JR (1980) The influence of codon context on genetic code translation. Nature 286:123–127
Buck M, Griffiths E (1981) Regulation of aromatic amino acid transport by tRNA: role of 2-methylthio-N6-(Δ 2-isopentenyl)-adenosine. Nucl Acids Res 9:401–414
Colby DS, Schedl P, Guthrie C (1976) A functional requirement for modification of the wobble nucleotide in the anticodon of a T4 suppressor tRNA. Cell 9:449–463
Dagert M, Ehrlich SD (1979) Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene 6:23–28
Feinstein SI, Altman S (1977) Coding properties of an ochre-suppressing derivative of Escherichia coli tRNA Tyr1 . J Mol Biol 112:453–470
Fluck MM, Salser W, Epstein RH (1977) The influence of the reading context upon the suppression of nonsense codons. Mol Gen Genet 151:137–149
Fluck MM, Epstein RH (1980) Isolation and characterization of context mutations affecting the suppressibility of nonsense mutations. Mol Gen Genet 177:615–627
Gauss DH, Sprinzl M (1983) Compilation of tRNA sequences. Nucl Acids Res 11:r1-r54
Gorini L (1974) Streptomycin and misreading of the genetic code. In: Nomura M, Tissières A, Lengyel P (eds) Ribosomes. Cold Spring Harbor Laboratory, New York, pp 791–803
Hagervall TG, Björk GR (1984) Genetic mapping and cloning of the gene (trmC) responsible for the synthesis of tRNA (mnm5s2U)methyltransferase in Escherichia coli. Mol Gen Genet 196:201–207
Hillen W, Egert E, Lindner HJ, Gassen HG (1978) Restriction or amplification of wobble recognition: The structure of 2-thio-5-methyl-aminomethyluridine and the interaction of odd uridines with the anticodon dloop backbone. FEBS Lett 94:361–364
Janner F, Vögeli G, Fluri R (1980) The antisuppressor strain sin1 of Schizosaccharomyces pombe lacks the modification isopentenyl-adenosine in transfer RNA. J Mol Biol 139:207–219
Johnson PF, Abelson J (1983) The yeast tRNAtyr gene intron is essential for correct modification if its tRNA product. Nature 302:681–687
Johnston HM, Barnes WM, Chunley FG, Bossi L, Roth JR (1980) Model for regulation of the histidine operon of Salmonella. Proc Natl Acad Sci USA 77:508–512
Kern D, Lapointe J (1979) Glutamyl transfer ribonucleic acid synthetase of Escherichia coli. Effect of alteration of the 5-(methylaminomethyl)-2-thiouridine in the anticodon of glutamic acid transfer ribonucleic acid on the catalytic mechanism. Biochemistry 18:5819–5826
Laten H, Gorman J, Bock RM (1978) Isopentenyladenosine deficient tRNA from an antisuppressor mutant of Saccharomyces cerevisiae. Nucl Acids Res 5:4329–4342
Marinus MG, Morris NR, Söll D, Kwong TC (1975) Isolation and partial characterization of three Escherichia coli mutants with altered transfer ribonucleic acid methylases. J Bacteriol 122:257–265
Miller JH, Ganem D, Lu P, Schmitz A (1977) Genetic studies of the lac repressor I. Correlation of mutational sites with specific amino acid residues: Construction of a colinear gene-protein map. J Mol Biol 109:275–301
Miller JH, Albertini AM (1983) Effects of surrounding sequence on the suppression of nonsense codons. J Mol Biol 164:59–71
Müller-Hill B, Kania J (1974) Lac repressor can be fused to β-galactosidase. Nature 249:561–562
Neidhardt FC, Block PL, Smith DF (1974) Culture medium for enterobacteria. J Bacteriol 119:736–747
Neidhardt FC, Bloch PL, Pedersen S, Reeh S (1977) Chemical measurement of steady-state levels of ten aminoacyl-transfer ribonucleic acid synthetases in Escherichia coli. J Bacteriol 129:378–387
Ohashi Z, Saneyoshi M, Harada F, Hara H, Nishimura S (1970) Presumed anticodon structure of glutamic acid tRNA from E. coli: A possible location of a 2-thiouridine derivative in the first position of the anticodon. Biochem Biophys Res Commun 40:866–872
Petrullo LA, Gallagher PJ, Elseviers D (1983) The role of 2-methylthio-N6-isopentenyladenosine in readthrough and suppression of nonsense codons in Escherichia coli. Mol Gen Genet 190:289–294
Prather NE, Mims BH, Murgola EJ (1983) supG and supL in Escherichia coli code for mutant lysine tRNAs. Nucl Acid Res 11:8283–8286
Rogg H, Brambilla R, Keith G, Staehelin M (1975) An improved method for the separation and quantitation of the modified nucleosides of transfer RNA. Nucl Acids Res 1:285–295
Salser W (1969) The influence of the reading context upon the suppression of nonsense codons. Mol Gen Genet 105:125–130
Salser W, Fluck MM, Epstein RH (1969) The influence of the reading context upon the suppression of nonsense codons. Cold Spring Harbor Symp Quant Biol 34:513–520
Sekiya T, Takeishi K, Ukita U (1969) Specificity of yeast glutamic acid transfer RNA for codon recognition. Biochim Biophys Acta 182:411–426
Sherman R, Ono B, Stewart JR (1979) Use of the iso-l-cytochrome C system for investigating nonsense mutants and suppressors in yeast. In: Celis JE, Smith JD (eds) Nonsense mutations and tRNA suppressors. Academic Press, London, pp 133–153
Smith JD (1979) Suppressor tRNAs in procaryotes. In: Celis JE, Smith JD (eds) Nonsense mutations and tRNA suppressors. Academic Press, London, pp 109–125
Vogel HJ, Bonner DM (1956) Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem 218:97–106
Waldron C, Cox BS, Wills N, Gesteland RF, Piper PW, Colby D, Guthrie C (1981) Yeast ochre suppressors SUQ5-ol is an altered tRNA serUCA . Nucl Acids Res 9:3077–3088
Weissenbach J, Dirheimer G (1978) Paring properties of the methylester of 5-carboxymethyl uridine in the wobble position of yeast trna Arg3 . Biochim Biophys Acta 518:530–534
Yanofsky C, Soll L (1977) Mutations affecting tRNAtrp and its charging and their effect on regulation of transcription termination at the attenuator of the tryptophan operon. J Mol Biol 113:663–677
Yahata H, Ocada Y, Tsugita A (1970) Adjacent effect on suppression efficiency. II. Study on ochre and amber mutants of T4 phage lysozyme. Mol Gen Genet 106:208–212
Author information
Authors and Affiliations
Additional information
Communicated by A. Böck
Rights and permissions
About this article
Cite this article
Hagervall, T.G., Björk, G.R. Undermodification in the first position of the anticodon of supG-tRNA reduces translational efficiency. Mol Gen Genet 196, 194–200 (1984). https://doi.org/10.1007/BF00328050
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00328050