Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1991 Jul 11;19(13):3499–3506. doi: 10.1093/nar/19.13.3499

Distinctive patterns of translational reinitiation in the lac repressor mRNA: bridging of long distances by out-of-frame translation and RNA secondary structure, effects of primary sequence.

R J Matteson 1, S J Biswas 1, D A Steege 1
PMCID: PMC328371  PMID: 1906601

Abstract

In the early region of the Escherichia coli lac repressor mRNA, translational reinitiation events triggered by nonsense codons occur over long distances and in a distinctive pattern not explained by simple use of the next available initiator triplet. Defined fusions of the restart sites to the lacZ coding region have been used to explore the basis for these reinitiation patterns and to ask whether the sites can function in independent initiation at the 5' end of an mRNA. The results obtained confirm earlier indications that the restart sites may have little or no inherent capacity for binding free 30S ribosomes. The data also add to growing evidence that primary sequence elements are important determinants of reinitiation efficiency. On the basis of the reinitiation activities for nonsense sites throughout the early region of the mRNA, we suggest that out-of-frame restarts and RNA secondary structure bridge long distances between the point of termination and downstream restart codons. Such bridging mechanisms could serve more generally as a means of propagating translational activity across long polycistronic mRNAs.

Full text

PDF
3503

Selected References

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

  1. Berkhout B., van Duin J. Mechanism of translational coupling between coat protein and replicase genes of RNA bacteriophage MS2. Nucleic Acids Res. 1985 Oct 11;13(19):6955–6967. doi: 10.1093/nar/13.19.6955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Calos M. P. DNA sequence for a low-level promoter of the lac repressor gene and an 'up' promoter mutation. Nature. 1978 Aug 24;274(5673):762–765. doi: 10.1038/274762a0. [DOI] [PubMed] [Google Scholar]
  3. Casadaban M. J., Martinez-Arias A., Shapira S. K., Chou J. Beta-galactosidase gene fusions for analyzing gene expression in escherichia coli and yeast. Methods Enzymol. 1983;100:293–308. doi: 10.1016/0076-6879(83)00063-4. [DOI] [PubMed] [Google Scholar]
  4. Cech T. R., Tanner N. K., Tinoco I., Jr, Weir B. R., Zuker M., Perlman P. S. Secondary structure of the Tetrahymena ribosomal RNA intervening sequence: structural homology with fungal mitochondrial intervening sequences. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3903–3907. doi: 10.1073/pnas.80.13.3903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cone K. C., Sellitti M. A., Steege D. A. Lac repressor mRNA transcription terminates in vivo in the lac control region. J Biol Chem. 1983 Sep 25;258(18):11296–11304. [PubMed] [Google Scholar]
  6. Cone K. C., Steege D. A. Functional analysis of lac repressor restart sites in translational initiation and reinitiation. J Mol Biol. 1985 Dec 20;186(4):733–742. doi: 10.1016/0022-2836(85)90393-6. [DOI] [PubMed] [Google Scholar]
  7. Cone K. C., Steege D. A. Messenger RNA conformation and ribosome selection of translational reinitiation sites in the lac repressor mRNA. J Mol Biol. 1985 Dec 20;186(4):725–732. doi: 10.1016/0022-2836(85)90392-4. [DOI] [PubMed] [Google Scholar]
  8. Das A., Yanofsky C. A ribosome binding site sequence is necessary for efficient expression of the distal gene of a translationally-coupled gene pair. Nucleic Acids Res. 1984 Jun 11;12(11):4757–4768. doi: 10.1093/nar/12.11.4757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Farabaugh P. J. Sequence of the lacI gene. Nature. 1978 Aug 24;274(5673):765–769. doi: 10.1038/274765a0. [DOI] [PubMed] [Google Scholar]
  10. Files J. G., Weber K., Miller J. H. Translational reinitiation: reinitiation of lac repressor fragments at three internal sites early in the lac i gene of Escherichia coli. Proc Natl Acad Sci U S A. 1974 Mar;71(3):667–670. doi: 10.1073/pnas.71.3.667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ganem D., Miller J. H., Files J. G., Platt T., Weber K. Reinitiation of a lac repressor fragment at a codon other than AUG. Proc Natl Acad Sci U S A. 1973 Nov;70(11):3165–3169. doi: 10.1073/pnas.70.11.3165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  13. Hartz D., McPheeters D. S., Traut R., Gold L. Extension inhibition analysis of translation initiation complexes. Methods Enzymol. 1988;164:419–425. doi: 10.1016/s0076-6879(88)64058-4. [DOI] [PubMed] [Google Scholar]
  14. Huang W. M., Ao S. Z., Casjens S., Orlandi R., Zeikus R., Weiss R., Winge D., Fang M. A persistent untranslated sequence within bacteriophage T4 DNA topoisomerase gene 60. Science. 1988 Feb 26;239(4843):1005–1012. doi: 10.1126/science.2830666. [DOI] [PubMed] [Google Scholar]
  15. Ivey-Hoyle M., Steege D. A. Translation of phage f1 gene VII occurs from an inherently defective initiation site made functional by coupling. J Mol Biol. 1989 Jul 20;208(2):233–244. doi: 10.1016/0022-2836(89)90385-9. [DOI] [PubMed] [Google Scholar]
  16. Martin J., Webster R. E. The in vitro translation of a terminating signal by a single Escherichia coli ribosome. The fate of the subunits. J Biol Chem. 1975 Oct 25;250(20):8132–8139. [PubMed] [Google Scholar]
  17. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  18. Miller J. H., Ganem D., Lu P., Schmitz A. 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. 1977 Jan 15;109(2):275–298. doi: 10.1016/s0022-2836(77)80034-x. [DOI] [PubMed] [Google Scholar]
  19. Napoli C., Gold L., Singer B. S. Translational reinitiation in the rIIB cistron of bacteriophage T4. J Mol Biol. 1981 Jul 5;149(3):433–449. doi: 10.1016/0022-2836(81)90480-0. [DOI] [PubMed] [Google Scholar]
  20. Platt T., Weber K., Ganem D., Miller J. H. Translational restarts: AUG reinitiation of a lac repressor fragment. Proc Natl Acad Sci U S A. 1972 Apr;69(4):897–901. doi: 10.1073/pnas.69.4.897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ross G. W., O'Callaghan C. H. Beta-lactamase assays. Methods Enzymol. 1975;43:69–85. doi: 10.1016/0076-6879(75)43081-6. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Sarabhai A., Brenner S. A mutant which reinitiates the polypeptide chain after chain termination. J Mol Biol. 1967 Jul 14;27(1):145–162. doi: 10.1016/0022-2836(67)90357-9. [DOI] [PubMed] [Google Scholar]
  24. Schmidt B. F., Berkhout B., Overbeek G. P., van Strien A., van Duin J. Determination of the RNA secondary structure that regulates lysis gene expression in bacteriophage MS2. J Mol Biol. 1987 Jun 5;195(3):505–516. doi: 10.1016/0022-2836(87)90179-3. [DOI] [PubMed] [Google Scholar]
  25. Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. doi: 10.1073/pnas.71.4.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Spanjaard R. A., van Duin J. Translational reinitiation in the presence and absence of a Shine and Dalgarno sequence. Nucleic Acids Res. 1989 Jul 25;17(14):5501–5507. doi: 10.1093/nar/17.14.5501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Steege D. A. 5'-Terminal nucleotide sequence of Escherichia coli lactose repressor mRNA: features of translational initiation and reinitiation sites. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4163–4167. doi: 10.1073/pnas.74.10.4163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Steitz J. A. Polypeptide chain initiation: nucleotide sequences of the three ribosomal binding sites in bacteriophage R17 RNA. Nature. 1969 Dec 6;224(5223):957–964. doi: 10.1038/224957a0. [DOI] [PubMed] [Google Scholar]
  29. Turner D. H., Sugimoto N., Freier S. M. RNA structure prediction. Annu Rev Biophys Biophys Chem. 1988;17:167–192. doi: 10.1146/annurev.bb.17.060188.001123. [DOI] [PubMed] [Google Scholar]
  30. Weiss R. B., Dunn D. M., Atkins J. F., Gesteland R. F. Slippery runs, shifty stops, backward steps, and forward hops: -2, -1, +1, +2, +5, and +6 ribosomal frameshifting. Cold Spring Harb Symp Quant Biol. 1987;52:687–693. doi: 10.1101/sqb.1987.052.01.078. [DOI] [PubMed] [Google Scholar]
  31. Weiss R. B., Dunn D. M., Dahlberg A. E., Atkins J. F., Gesteland R. F. Reading frame switch caused by base-pair formation between the 3' end of 16S rRNA and the mRNA during elongation of protein synthesis in Escherichia coli. EMBO J. 1988 May;7(5):1503–1507. doi: 10.1002/j.1460-2075.1988.tb02969.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Young R. A., Davis R. W. Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1194–1198. doi: 10.1073/pnas.80.5.1194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Zuker M. On finding all suboptimal foldings of an RNA molecule. Science. 1989 Apr 7;244(4900):48–52. doi: 10.1126/science.2468181. [DOI] [PubMed] [Google Scholar]
  34. Zuker M., Stiegler P. Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res. 1981 Jan 10;9(1):133–148. doi: 10.1093/nar/9.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES