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. 1985 Feb 11;13(3):893–909. doi: 10.1093/nar/13.3.893

Translationally coupled initiation of protein synthesis in Bacillus subtilis.

R Sprengel, B Reiss, H Schaller
PMCID: PMC341041  PMID: 3923434

Abstract

The neomycin phosphotransferase gene (neo) from Transposon Tn5 is active in Gram-negative bacteria but silent in B. subtilis since it lacks an appropriate ribosome binding site for Gram-positive bacteria. Neo translation could be reactivated by coupling its initiation to the translational termination of the highly expressed beta-lactamase gene (penP) from B. licheniformis. This initiation occurred at the authentic neo start codon. Its efficiency was independent of the nucleotide sequence 5 to the neo gene, but strongly affected by the distance between the termination and initiation codon. It was the highest if both codons overlapped in the sequence ATGA. In B. licheniformis, a translationally coupled neo gene was inducible expressed as the penP gene demonstrating the potential of the technique to monitor the activity of expression units for which no direct assays exists.

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Selected References

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

  1. Backman K., Ptashne M., Gilbert W. Construction of plasmids carrying the cI gene of bacteriophage lambda. Proc Natl Acad Sci U S A. 1976 Nov;73(11):4174–4178. doi: 10.1073/pnas.73.11.4174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beck E., Ludwig G., Auerswald E. A., Reiss B., Schaller H. Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene. 1982 Oct;19(3):327–336. doi: 10.1016/0378-1119(82)90023-3. [DOI] [PubMed] [Google Scholar]
  3. Berg D. E., Davies J., Allet B., Rochaix J. D. Transposition of R factor genes to bacteriophage lambda. Proc Natl Acad Sci U S A. 1975 Sep;72(9):3628–3632. doi: 10.1073/pnas.72.9.3628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bremer E., Cole S. T., Hindennach I., Henning U., Beck E., Kurz C., Schaller H. Export of a protein into the outer membrane of Escherichia coli K12. Stable incorporation of the OmpA protein requires less than 193 amino-terminal amino-acid residues. Eur J Biochem. 1982 Feb;122(1):223–231. doi: 10.1111/j.1432-1033.1982.tb05870.x. [DOI] [PubMed] [Google Scholar]
  6. Contente S., Dubnau D. Marker rescue transformation by linear plasmid DNA in Bacillus subtilis. Plasmid. 1979 Oct;2(4):555–571. doi: 10.1016/0147-619x(79)90054-4. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Dixon L. K., Hohn T. Initiation of translation of the cauliflower mosaic virus genome from a polycistronic mRNA: evidence from deletion mutagenesis. EMBO J. 1984 Dec 1;3(12):2731–2736. doi: 10.1002/j.1460-2075.1984.tb02203.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dubnau D. A., Pollock M. R. The genetics of Bacillus licheniformis penicillinase: a preliminary analysis from studies on mutation and inter-strain and intra-strain transformations. J Gen Microbiol. 1965 Oct;41(1):7–21. doi: 10.1099/00221287-41-1-7. [DOI] [PubMed] [Google Scholar]
  10. Duncan C. H., Wilson G. A., Young F. E. Mechanism of integrating foreign DNA during transformation of Bacillus subtilis. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3664–3668. doi: 10.1073/pnas.75.8.3664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Emr S. D., Hall M. N., Silhavy T. J. A mechanism of protein localization: the signal hypothesis and bacteria. J Cell Biol. 1980 Sep;86(3):701–711. doi: 10.1083/jcb.86.3.701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Gheysen D., Iserentant D., Derom C., Fiers W. Systematic alteration of the nucleotide sequence preceding the translation initiation codon and the effects on bacterial expression of the cloned SV40 small-t antigen gene. Gene. 1982 Jan;17(1):55–63. doi: 10.1016/0378-1119(82)90100-7. [DOI] [PubMed] [Google Scholar]
  14. Gray O., Chang S. Molecular cloning and expression of Bacillus licheniformis beta-lactamase gene in Escherichia coli and Bacillus subtilis. J Bacteriol. 1981 Jan;145(1):422–428. doi: 10.1128/jb.145.1.422-428.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gryczan T. J., Dubnau D. Construction and properties of chimeric plasmids in Bacillus subtilis. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1428–1432. doi: 10.1073/pnas.75.3.1428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Herrmann R., Neugebauer K., Pirkl E., Zentgraf H., Schaller H. Conversion of bacteriophage fd into an efficient single-stranded DNA vector system. Mol Gen Genet. 1980 Jan;177(2):231–242. doi: 10.1007/BF00267434. [DOI] [PubMed] [Google Scholar]
  17. Iglesias A., Bensi G., Canosi U., Trautner T. A. Plasmid transformation in Bacillus subtilis. Alterations introduced into the recipient-homologous DNA of hybrid plasmids can be corrected in transformation. Mol Gen Genet. 1981;184(3):405–409. doi: 10.1007/BF00352513. [DOI] [PubMed] [Google Scholar]
  18. Imanaka T., Tanaka T., Tsunekawa H., Aiba S. Cloning of the genes for penicillinase, penP and penI, of Bacillus licheniformis in some vector plasmids and their expression in Escherichia coli, Bacillus subtilis, and Bacillus licheniformis. J Bacteriol. 1981 Sep;147(3):776–786. doi: 10.1128/jb.147.3.776-786.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Iserentant D., Fiers W. Secondary structure of mRNA and efficiency of translation initiation. Gene. 1980 Apr;9(1-2):1–12. doi: 10.1016/0378-1119(80)90163-8. [DOI] [PubMed] [Google Scholar]
  20. Kozak M. Selection of initiation sites by eucaryotic ribosomes: effect of inserting AUG triplets upstream from the coding sequence for preproinsulin. Nucleic Acids Res. 1984 May 11;12(9):3873–3893. doi: 10.1093/nar/12.9.3873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kreft J., Berger H., Härtlein M., Müller B., Weidinger G., Goebel W. Cloning and expression in Escherichia coli and Bacillus subtilis of the hemolysin (cereolysin) determinant from Bacillus cereus. J Bacteriol. 1983 Aug;155(2):681–689. doi: 10.1128/jb.155.2.681-689.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. McLaughlin J. R., Murray C. L., Rabinowitz J. C. Unique features in the ribosome binding site sequence of the gram-positive Staphylococcus aureus beta-lactamase gene. J Biol Chem. 1981 Nov 10;256(21):11283–11291. [PubMed] [Google Scholar]
  24. Moran C. P., Jr, Lang N., LeGrice S. F., Lee G., Stephens M., Sonenshein A. L., Pero J., Losick R. Nucleotide sequences that signal the initiation of transcription and translation in Bacillus subtilis. Mol Gen Genet. 1982;186(3):339–346. doi: 10.1007/BF00729452. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Neugebauer K., Sprengel R., Schaller H. Penicillinase from Bacillus licheniformis: nucleotide sequence of the gene and implications for the biosynthesis of a secretory protein in a Gram-positive bacterium. Nucleic Acids Res. 1981 Jun 11;9(11):2577–2588. doi: 10.1093/nar/9.11.2577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Reiss B., Sprengel R., Will H., Schaller H. A new sensitive method for qualitative and quantitative assay of neomycin phosphotransferase in crude cell extracts. Gene. 1984 Oct;30(1-3):211–217. doi: 10.1016/0378-1119(84)90122-7. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Schümperli D., McKenney K., Sobieski D. A., Rosenberg M. Translational coupling at an intercistronic boundary of the Escherichia coli galactose operon. Cell. 1982 Oct;30(3):865–871. doi: 10.1016/0092-8674(82)90291-4. [DOI] [PubMed] [Google Scholar]
  30. Sherratt D. J., Collins J. F. Analysis by transformation of the penicillinase system in Bacillus licheniformis. J Gen Microbiol. 1973 May;76(1):217–230. doi: 10.1099/00221287-76-1-217. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  33. 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]
  34. Thorne C. B., Stull H. B. Factors affecting transformation of Bacillus licheniformis. J Bacteriol. 1966 Mar;91(3):1012–1020. doi: 10.1128/jb.91.3.1012-1020.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Zabeau M., Stanley K. K. Enhanced expression of cro-beta-galactosidase fusion proteins under the control of the PR promoter of bacteriophage lambda. EMBO J. 1982;1(10):1217–1224. doi: 10.1002/j.1460-2075.1982.tb00016.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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