Summary
The expression of infC, the structural gene for translational initiation factor IF3, has been studied in different constructs under the control of the λPL and tac promoters. The amount of synthesized IF3 has been determined by a quantitative functional test and the levels of IF3-specific mRNA have been estimated. The synthesis of IF3 is strongly enhanced when the unusual AUU initiation codon is changed to AUG by site-directed mutagenesis. Removal of the sequence upstream from the start codon including most of the Shine-Dalgarno sequence, as well as part of a 10 bp region with potential complementarity to an internal region of the 16S rRNA, which is unique to the IF3 mRNA, reduced but did not completely abolish the high expression of infC obtained after introduction of the AUG initiation codon. The level of IF3 mRNA was found to be positively influenced by the presence of the rplT gene in the plasmid downstream from the infC gene. In vivo accumulation of a large excess of IF3, obtained when the infC gene was placed under the control of an incompletely repressed tac promoter, was not accompanied by any noticeable adverse phenotype.
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References
Bernard HU, Remaut E, Hershfield MV, Das HK, Helinski DR, Yanofsky C, Franklin N (1979) Construction of plasmid cloning vehicles that promote gene expression from the bacteriophage lambda PL promoter. Gene 5:59–76
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening of recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523
Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heynecker HL, Boyer HW, Crosa JH, Falkow, S (1977) Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95–113
Brombach M, Gualerzi C, Nakamura Y, Pon CL (1986) Molecular cloning and sequence of the Bacillus stearothermophilus initiation factor IF2 gene. Mol Gen Genet 205:97–102
Brosius J, Holy A (1984) Regulation of ribosomal RNA promoters with a synthetic lac operator. Proc Natl Acad Sci USA 81:6929–6933
Chang ACY, Cohen SN (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 134:1141–1156
Coleman J, Green PJ, Inouye M (1984) The use of RNAs complementary to specific mRNAs to regulate the expression of individual bacterial genes. Cell 37:429–436
De Boer HA, Hui A, Comstock LJ, Wong E, Vasser M (1983) Portable Shine-Dalgarno regions: a system for a systematic study of defined alterations of nucleotide sequences within E. coli ribosome binding sites. DNA 2:231–235
Doerper T, Winnacker E-L (1982), Improvements in the phosphoramidite procedure for the synthesis of oligodeoxyribonucleotides. Nucleic Acids Res 11:2575–2584
Elhardt D, Wirth R, Böck A (1982) Regulation of formation of threonyl-tRNA synthetase, phenylalanyl-tRNA synthetase and protein synthesis initiation factor 3 from E. coli in vivo and in vitro. Eur J Biochem 123:477–482
Fayat GF, Mayaux JF, Sacerdot C, Fromant M, Springer M, Grunberg-Manago M, Blanquet S (1983) E. coli phenylalanyl-tRNA synthetase operon region. Evidence for an attenuation mechanism. Identification of the gene for the ribosomal protein L20. J Mol Biol 171:239–261
Gold L, Stormo G, Saunders R (1984) Escherichia coli translational initiation factor IF3: a unique case of translational regulation. Proc Natl Acad Sci USA 81:7061–7065
Grunberg-Manago M, Plumbridge JA, Sacerdot C, Howe JG, Springer M, Hershey JWB, Mayaux JF, Fayat G, Blanquet S (1984) The organisation and expression of genes for translational initiation factors. In: Clark BFC, Petersen HU (eds), Gene Expression. Munksgaard, Copenhagen, pp 22–37
Gualerzi C, Pon CL (1981) Protein biosynthesis in prokaryotic cells: mechanism of 30S initiation complex formation in E. coli. In: Balaban M, Sussman JL, Traub G, Yonath A (eds) Structural aspects of recognition and assembly in biological macromolecules, Vol 2. Balaban ISS, Rehovot and Philadelphia, pp 805–826
Gualerzi CO, Pon CL, Pawlik RT, Canonaco MA, Paci M, Wintermeyer W (1986) Role of the initiation factors in E. coli translational initiation. In: Hardesty B, Kramer G (eds) Ribosomes — Structure, Function and Genetics, Springer, New York, pp 621–641
Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580
Hennecke H, Springer M, Böck A (1977) A specialized transducing λ phage carrying the Escherichia coli genes for phenylalanyl-tRNA synthetase. Mol Gen Genet 152:205–210
Kramer W, Drutsa V, Jansen H-W, Kramer B, Pflugfelder M, Fritz H-J (1984) The gapped duplex, DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res 12:9441–9456
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular Cloning. a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Matteucci MD, Caruthers MH (1981) Synthesis of deoxyoligonucleotides on a polymer support. J Amer Chem Soc 103:3185–3191
Messing J, Crea R, Seeburg PH (1981) A system for shotgun DNA sequencing. Nucleic Acids Res 9:309–321
Miller JH (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory. Cold Spring Harbor, New York
O'Farrell PH, Kutter E, Nakanishi M (1980) A restriction map of the bacteriophage T4 genome. Mol Gen Genet 179:421–435
Ohsawa H, Herrlich P, Gualerzi C (1984) In vitro template activity of 0.3 mRNA from wild type and initiation mutants of bacteriophage T7. Mol Gen Genet 196:53–58
Pawlik RT, Littlechild J, Pon C Gualerzi C (1981) Purification and properties of Escherichia coli translational initiation factors. Biochem Int 2:421–428
Pon C, Gualerzi C (1979) Qualitative, and semiquantitative, assay of Escherichia coli translational initiation factor IF3. Methods Enzymol 60:230–239
Pon CL, Gualerzi CO (1986) Mechanism, of translational initiation in prokaryotes. IF3 is released from ribosomes during and not before 70S initiation complex formation. FEBS Lett 195:215–219
Remaut E, Stanssens P, Fiers W (1981) Plasmid vectors for high-efficiency expression controlled by the pL promoter of the coliphage lambda. Gene 15:81–93
Remaut E, Tsao H, Fiers W (1983) Improved plasmid vectors with a thermoinducible expression and temperature-regulated runaway replication. Gene 22:103–113
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Shine J, Dalgarno L (1974) The 3′-terminal sequence of Escherichia coli 16S ribosomal RNA: Complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci USA 71:1342–1346
Smith AJH (1980) DNA sequence analysis by primed synthesis. Methods Enzymol 65:560–580
Springer M, Graffe M, Hennecke H (1977) Specialized transducing phage for the initiation factor 3 gene in Escherichia coli. Proc Natl Acad Sci USA 74:3970–3974
Stueber D, Bujard H (1982) Transcription from efficient promoters can interfere with plasmid replication and diminish expression of plasmid specified genes. EMBO J 1:1399–1404
Thomas PS (1983) Hybridization of denatured RNA transferred or dotted to nitrocellulose paper. Methods Enzymol 100:255–266
Tullis RH, Rubin H (1980) Calcium protects DNase I from proteinase K; a new method for the removal of contaminating RNase from DNase I. Anal Biochem 107:260–264
Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: Nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119
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Brombach, M., Pon, C.L. The unusual translational initiation codon AUU limits the expression of the infC (initiation factor IF3) gene of Escherichia coli . Mole Gen Genet 208, 94–100 (1987). https://doi.org/10.1007/BF00330428
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DOI: https://doi.org/10.1007/BF00330428