Molecular and General Genetics MGG

, Volume 235, Issue 2–3, pp 325–332 | Cite as

DNA sequences required for translational frameshifting in production of the transposase encoded by IS 1

  • Yasuhiko Sekine
  • Eiichi Ohtsubo
Original Articles


The transposase encoded by insertion sequence IS 1 is produced from two out-of-phase reading frames (insA and B′-insB) by translational frameshifting, which occurs within a run of six adenines in the −1 direction. To determine the sequence essential for frameshifting, substitution mutations were introduced within the region containing the run of adenines and were examined for their effects on frameshifting. Substitutions at each of three (2nd, 3rd and 4th) adenine residues in the run, which are recognized by tRNALys reading insA, caused serious defects in frameshifting, showing that the three adenine residues are essential for frameshifting. The effects of substitution mutations introduced in the region flanking the run of adenines and in the secondary structures located downstream were, however, small, indicating that such a region and structures are not essential for frameshifting. Deletion of a region containing the termination codon of insA caused a decrease in β-galactosidase activity specified by the lacZ fusion plasmid in frame with B′-insB. Exchange of the wild-type termination codon of insA for a different one or introduction of an additional termination codon in the region upstream of the native termination codon caused an increase in β-galactosidase activity, indicating that the termination codon in insA affects the efficiency of frameshifting.

Key words

Adenine run Cointegration Secondary structure of mRNA Termination codon tRNALys 


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  1. Atkins JF, Weiss RB, Gesteland RF (1990) Ribosome gymnastics — Degree of difficulty 9.5, style 10.0. Cell 62:413–423Google Scholar
  2. Brierley I, Digard P, Inglis SC (1989) Characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an RNA pseudoknot. Cell 57:537–547Google Scholar
  3. Casadaban MJ, Cohen SN (1980) Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol 138:179–207Google Scholar
  4. Chakraburtty K, Steinschneider A, Case RV, Mehler AH (1975) Primary structure of tRNALys of E. coli B. Nucleic Acids Res 2:2069–2075Google Scholar
  5. Dinman JD, Icho T, Wickner RB (1991) A −1 ribosomal frameshift in a double-stranded RNA virus of yeast forms a gag-pol fusion protein. Proc Natl Acad Sci USA 88:174–178Google Scholar
  6. Flower AM, McHenry CS (1990) The γ subunit of DNA polymerase III holoenzyme of Escherichia coli is produced by ribosomal frameshifting. Proc Natl Acad Sci USA 87:3713–3717Google Scholar
  7. Hashimoto-Gotoh T, Sekiguchi M (1977) Mutations to temperature sensitivity in R plasmid pSC101. J Bacteriol 131:405–412Google Scholar
  8. Hübner P, Iida S, Arber W (1987) A transcriptional terminator sequence in the prokaryotic transposable element IS1. Mol Gen Genet 206:485–490Google Scholar
  9. Jacks T, Townsley K, Varmus HE, Majors J (1987) Two efficient ribosomal frameshifting events are required for synthesis of mouse mammary virus gag-related polyprotein. Proc Natl Acad Sci USA 82:2829–2833Google Scholar
  10. Jacks T, Madhani HD, Masiarz FR, Varmus HE (1988) Signals for ribosomal frameshifting in the Rous sarcoma virus gag-pol region. Cell 55:447–458Google Scholar
  11. Kunkel TA, Roberts JD, Zakour RA (1987) Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol 154:367–382Google Scholar
  12. Machida Y, Machida C, Ohtsubo E (1982) A novel type of transposon generated by insertion element IS102 present in a pSC101 derivative. Cell 30:29–36Google Scholar
  13. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular Cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  14. Messing J (1983) New M13 vectors for cloning. Methods Enzymol 101:20–78Google Scholar
  15. Ohtsubo E, Zenilman M, Ohtsubo H, McCormick M, Machida C, Machida Y (1981) Mechanism of insertion and cointegration mediated by IS1 and Tn3. Cold Spring Harbor Symp Quant Biol 45:283–295Google Scholar
  16. Ohtsubo H, Obtsubo E (1978) Nucleotide sequence of an insertion element, IS1. Proc Natl Acad Sci USA 73:2316–2320Google Scholar
  17. Oppenheim D, Yanofsky C (1980) Translational coupling during expression of the tryptophan operon of Escherichia coli. Genetics 95:785–795Google Scholar
  18. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467Google Scholar
  19. Scheit KH, Faerber P (1975) The effects of thioketo substitution upon uracil-adenine interactions in polyribonucleotides. Eur J Biochem 50:549–555Google Scholar
  20. Sekine Y, Ohtsubo E (1989) Frameshifting is required for production of the transposase encoded by insertion sequence 1. Proc Natl Acad Sci USA 86:4609–4613Google Scholar
  21. Sekine Y, Ohtsubo E (1991) Translational frameshifting in IS elements and other genetic systems. In: Kimura M, Takahata N (ed) New Aspects of The Genetics of Molecular Evolution. Japan Scientific Societies Press, Tokyo/Springer-Verlag, Berlin, pp 243–261Google Scholar
  22. Sekine Y, Nagasawa H, Ohtsubo E (1992) Identification of the site of translational frameshifting required for production of the transposase encoded by insertion sequence IS1. Mol Gen Genet 235:317–324Google Scholar
  23. Tinoco I Jr, Borer PN, Dengler B, Levine MD, Uhlenbeck OC, Crothers DM, Gralla J (1973) Improved estimation of secondary structure in ribonucleic acids. Nature New Biol 246:40–41Google Scholar
  24. Tsuchihashi Z, Kornberg A (1990) Translational frameshifting generates the γ subunit of DNA polymerase III holoenzyme. Proc Natl Acad Sci USA 87:2516–2520Google Scholar
  25. Vieira J, Messing J (1987) Production of single stranded plasmid DNA. Methods Enzymol 153:3–11Google Scholar
  26. Vögele K, Schwartz E, Welz C, Schiltz E, Rak B (1991) High-level ribosomal frameshifting directs the synthesis of IS150 gene products. Nucleic Acids Res 19:4377–4385Google Scholar
  27. Weiss RB (1984) Molecular model of ribosome frameshifting. Proc Nail Acad Sci USA 81:5797–5801Google Scholar
  28. Weiss RB, Dunn DM, Atkins JF, Gesteland RF (1987) Slippery runs, shifty stops, backward steps, and forward hops: −2, −1, +1, +2, +5, and +6 ribosomal frameshifting. Cold Spring Harbor Symp Quant Biol 52:687–693Google Scholar
  29. Yokoyama S, Watanabe T, Murao K, Ishikura H, Yamaizumi Z, Nishimura S, Miyazawa T (1985) Molecular mechanism of codon recognition by tRNA species with modified uridine in the first position of the anticodon. Proc Nail Acad Sci USA 82:4905–4909Google Scholar
  30. Yoshioka Y, Ohtsubo H, Ohtsubo E (1987) Repressor gene finO in plasmids R100 and F: Constitutive transfer of plasmid F is caused by insertion of IS3 into F finO. J Bacteriol 169:619–623Google Scholar

Copyright information

© Springer-Verlag 1992

Authors and Affiliations

  • Yasuhiko Sekine
    • 1
  • Eiichi Ohtsubo
    • 1
  1. 1.Institute of Applied MicrobiologyUniversity of TokyoTokyoJapan

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