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Characterization of cis-acting mutations which increase expression of a glnS-lacZ fusion in Escherichia coli

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Summary

glnS-lacZ fusions have been used to isolate mutations which enhance expression of the glnS gene. One mutation, acting at the level of transcription changes the-10 region of the promoter from GATCAT to TATCAT and produces a ten-fold increase in mRNA. Four other mutations which enhance expression three-fold to nine-fold fall within the transcribed region, but not within the Shine and Dalgarno sequence nor in the initiator codon. These mutations are shown to enhance translation specifically and different models are considered to explain their mode of action.

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References

  • Altuvia S, Oppenheim AB (1986) Translational regulatory signals within the coding region of the bacteriophage λ cIII gene. J Bacteriol 167:415–419

    Google Scholar 

  • Bachmann BJ (1983) Linkage map of E. coli K12. Edition 7 Microbiol Rev 47:180–230

    Google Scholar 

  • Borck K, Beggs JD, Brammar WJ, Hopkins AS, Murray NE (1976) The construction in vitro of transducing derivatives of phage lambda. Mol Gen Genet 146:199–207

    Google Scholar 

  • Casadaban M, Chou J, Cohen SN (1980) In vitro gene fusions that join an enzymatically active β-galactosidase segment to amino-terminal fragments of exogenous proteins: E. coli plasmid vectors for the detection and cloning of translational initiation signals. J Bacteriol 143:971–980

    Google Scholar 

  • Chapon C (1982) Expression of malT, the regulator gene of the maltose operon in E. coli, is limited both at transcription and translation. EMBO J 1:369–374

    Google Scholar 

  • Cheung A, Söll D (1984) In vivo and in vitro transcription of the E. coli glutaminyl-tRNA synthetase gene. J Biol Chem 259:9953–9958

    Google Scholar 

  • Daldal F (1983) Molecular cloning of the gene for phosphofructokinase-2 of E. coli and the nature of a mutation pfkB1 causing a high level of the enzyme. J Mol Biol 168:285–305

    Google Scholar 

  • Davis RW, Botstein D, Roth JR (1980) Advanced Bacterial Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • Dreyfus M, Kotlarz D, Busby S (1985) Point mutations that affect translational initiation in the E. coli galE gene. J Mol Biol 182:411–417

    Google Scholar 

  • Gren EJ (1984) Recognition of mRNA during translational initiation in E. coli. Biochimie 66:1–29

    Google Scholar 

  • Grosjean H, Fiers W (1982) Preferential codon usage in prokaryotic genes: the optimal codon-anticodon interaction energy and the selective codon usage in efficiently expressed genes. Gene 18:199–209

    Google Scholar 

  • Hall MN, Gabay J, Débarbouillé M, Schwartz M (1982) A role for mRNA secondary structure in the control of translation initiation. Nature 295:616–618

    Google Scholar 

  • Hoben P, Uemura H, Yamao F, Cheung A, Swanson R, Summer-Smith M, Söll D (1984) Misaminoacylation by glutaminyl-tRNA synthetase: relaxed specificity in wild type and mutant enzymes. Fed Proc 43:2972–2976

    Google Scholar 

  • Hui A, Hayflick J, Dinkelspiel, de Boer HA (1984) Mutagenesis of the three bases preceding the start codon of the β-galactosidase mRNA and its affect on translation in E. coli. EMBO J 3:623–629

    Google Scholar 

  • Inokuchi H, Hoben P, Yamao F, Ozeki H, Söll D (1984) Transfer RNA mischarging mediated by a mutant E. coli glutaminyl-tRNA synthetase. Proc Natl Acad Sci USA 81:5076–5080

    Google Scholar 

  • Iserentant D, Fiers W (1980) Secondary structure of mRNA and efficiency of translation initiation. Gene 9:1–12

    Google Scholar 

  • Jacobson AB, Good L, Simonetti J, Zuker M (1984) Some simple computational methods to improve the folding of large RNAs. Nucl Acids Res 12:45–52

    Google Scholar 

  • Korner A, Magee BB, Liska B, Low KB, Adelberg EA, Söll D (1974) Isolation and partial characterisation of a temperature sensitive E. coli mutant with altered glutaminyl-tRNA synthetase. J Bacteriol 120:154–158

    Google Scholar 

  • Maniatis T, Fritsch E, Sambrook J (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • Marinus M (1987) DNA methylation in E. coli. Annu Rev Genet 21:113–131

    Google Scholar 

  • Matteucci MD, Heyneker HL (1983) Targeted random mutagenesis: the use of ambiguously synthesized oligonucleotides to mutagenize sequences immediately 5′ of an ATG initiation codon. Nucleic Acids Res 11:3113–3121

    Google Scholar 

  • Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • Neidhardt FC, Bloch PL, Smith DF (1974) Culture medium for enterobacteria. J Bacteriol 119:736–747

    Google Scholar 

  • Neidhardt FC, Bloch PL, Pedersen S, Reeh S (1977) Chemical measurement of steady-state levels of ten aminoacyl-tRNA synthetases in E. coli. J Bacteriol 129:378–387

    Google Scholar 

  • Place N, Fien K, Mahoney ME, Wulff D, Ho Y-S, Debouck C, Rosenberg M, Shih M-C, Gussin GN (1984) Mutations that alter the DNA binding site for the bacteriophage lambda cII protein and affect the translation efficiency of the cII gene. J Mol Biol 180:865–880

    Google Scholar 

  • Plumbridge JA (1987) The role of dam methylation in controlling gene expression. Biochimie 69:439–443

    Google Scholar 

  • Plumbridge JA, Söll D (1987) The effect of dam methylation on the expression of glnS in E. coli. Biochimie 69:539–541

    Google Scholar 

  • Plumbridge JA, Dondon J, Nakamura Y, Grunberg Manago M (1985) Effect of NusA protein on expression of the nusA, infB operon in E. coli. Nucl Acids Res 13:3371–3388

    Google Scholar 

  • Putney SD, Schimmel P (1981) An aminoacyl-tRNA synthetase binds to a specific DNA sequence and regulates its gene transcription. Nature 291:632–635

    Google Scholar 

  • Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    Google Scholar 

  • Scherer GFE, Walkinshaw MD, Arnott S, Morré DJ (1980) 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 8:3895–3907

    Google Scholar 

  • Schneider TD, Stormo GD, Gold L, Ehrenfeucht A (1986) Information content of binding sites on nucleotide sequences. J Mol Biol 188:415–431

    Google Scholar 

  • Shine J, Dalgarno L (1974) The 3′ terminal sequence of E. coli 16S rRNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci USA 71:1321–1346

    Google Scholar 

  • Shpaer EG (1986) Constraints on codon context in E. coli genes: their possible role in modulating the efficiency of translation. J Mol Biol 188:555–564

    Google Scholar 

  • Silhavy TJ, Berman ML, Enquist LW (1984) Experiments with gene fusions. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • Springer M, Trudel M, Graffe M, Plumbridge JA, Fayat G, Mayaux J-F, Sacerdot C, Blanquet S, Grunberg-Manago M (1983) E. coli phenylalanyl-tRNA synthetase operon is controlled by attenuation in vivo. J Mol Biol 171:263–279

    Google Scholar 

  • Springer M, Plumbridge JA, Butler JS, Graffe M, Dondon J, Mayaux J-F, Fayat G, Lestienne P, Blanquet S, Grunberg-Manago M (1985) Autogenous control of E. coli threonyl-tRNA synthetase expression in vivo. J Mol Biol 185:93–104

    Google Scholar 

  • Stanssens P, Remaut E, Fiers W (1985) Alterations upstream from the Shine-Dalgarno region and their effect on bacterial gene expression. Gene 36:211–223

    Google Scholar 

  • Stormo GD, Schneider TD, Gold LM (1982a) Characterization of translational initiation sites in E. coli. Nucleic Acids Res 10:2971–2996

    Google Scholar 

  • Stormo GD, Schneider TD, Gold LM, Ehrenfeucht A (1982b) Use of the “Perceptron” algorithm to distinguish translational initiation sites in E. coli. Nucleic Acids Res 10:2997–3011

    Google Scholar 

  • Swanson R, Hoben P, Summer-Smith M, Uemura H, Watson L, Söll D (1988) Accuracy of in vivo aminoacylation requires the proper balance of tRNA and aminoacyl-tRNA synthetase. Science 242:1548–1551

    Google Scholar 

  • Yamao F, Inokuchi H, Cheung A, Ozeki H, Söll D (1982) E. coli glutaminyl-tRNA synthetase. 1) Isolation and DNA sequence of the glnS gene. J Biol Chem 257:11639–11643

    Google Scholar 

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Communicated by H. Hennecke

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Plumbridge, J., Söll, D. Characterization of cis-acting mutations which increase expression of a glnS-lacZ fusion in Escherichia coli . Mol Gen Genet 216, 113–119 (1989). https://doi.org/10.1007/BF00332238

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