Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1994 Jul;176(13):4011–4016. doi: 10.1128/jb.176.13.4011-4016.1994

Mutational analysis of sequences in the recF gene of Escherichia coli K-12 that affect expression.

S J Sandler 1, A J Clark 1
PMCID: PMC205599  PMID: 8021183

Abstract

The level of translation of recF-lacZ fusions is reduced 20-fold by nucleotides 49 to 146 of recF. In this region of recF, we found a previously described ribosome-interactive sequence called epsilon and a hexapyrimidine tract located just upstream of the epsilon sequence. Mutational studies indicate that the hexapyrimidine sequence is involved in at least some of the reduced translation. When the hexapyrimidine sequence is mutant, mutating epsilon increases the level of translation maximally. We ruled out the possibility that ribosome frameshifting explains most of the effect of these two sequences on expression and suspect that multiple mechanisms may be responsible. In a separate report, we show that mutations in the hexapyrimidine tract and epsilon increase expression of the full-sized recF gene.

Full text

PDF
4011

Images in this article

4013Image
on p.4013

Selected References

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

  1. Atkins J. F., Weiss R. B., Gesteland R. F. Ribosome gymnastics--degree of difficulty 9.5, style 10.0. Cell. 1990 Aug 10;62(3):413–423. doi: 10.1016/0092-8674(90)90007-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bachmann B. J. Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol Rev. 1972 Dec;36(4):525–557. doi: 10.1128/br.36.4.525-557.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blanar M. A., Sandler S. J., Armengod M. E., Ream L. W., Clark A. J. Molecular analysis of the recF gene of Escherichia coli. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4622–4626. doi: 10.1073/pnas.81.15.4622. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brierley I., Digard P., Inglis S. C. Characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an RNA pseudoknot. Cell. 1989 May 19;57(4):537–547. doi: 10.1016/0092-8674(89)90124-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carter-Muenchau P., Wolf R. E., Jr Growth-rate-dependent regulation of 6-phosphogluconate dehydrogenase level mediated by an anti-Shine-Dalgarno sequence located within the Escherichia coli gnd structural gene. Proc Natl Acad Sci U S A. 1989 Feb;86(4):1138–1142. doi: 10.1073/pnas.86.4.1138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Falcone D., Andrews D. W. Both the 5' untranslated region and the sequences surrounding the start site contribute to efficient initiation of translation in vitro. Mol Cell Biol. 1991 May;11(5):2656–2664. doi: 10.1128/mcb.11.5.2656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gallie D. R., Kado C. I. A translational enhancer derived from tobacco mosaic virus is functionally equivalent to a Shine-Dalgarno sequence. Proc Natl Acad Sci U S A. 1989 Jan;86(1):129–132. doi: 10.1073/pnas.86.1.129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gesteland R. F., Weiss R. B., Atkins J. F. Recoding: reprogrammed genetic decoding. Science. 1992 Sep 18;257(5077):1640–1641. doi: 10.1126/science.1529352. [DOI] [PubMed] [Google Scholar]
  9. Gouy M., Gautier C. Codon usage in bacteria: correlation with gene expressivity. Nucleic Acids Res. 1982 Nov 25;10(22):7055–7074. doi: 10.1093/nar/10.22.7055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hartz D., McPheeters D. S., Green L., Gold L. Detection of Escherichia coli ribosome binding at translation initiation sites in the absence of tRNA. J Mol Biol. 1991 Mar 5;218(1):99–105. doi: 10.1016/0022-2836(91)90876-8. [DOI] [PubMed] [Google Scholar]
  11. Kozak M. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell. 1986 Jan 31;44(2):283–292. doi: 10.1016/0092-8674(86)90762-2. [DOI] [PubMed] [Google Scholar]
  12. Kozak M. Possible role of flanking nucleotides in recognition of the AUG initiator codon by eukaryotic ribosomes. Nucleic Acids Res. 1981 Oct 24;9(20):5233–5252. doi: 10.1093/nar/9.20.5233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lynch A. S., Wang J. C. Anchoring of DNA to the bacterial cytoplasmic membrane through cotranscriptional synthesis of polypeptides encoding membrane proteins or proteins for export: a mechanism of plasmid hypernegative supercoiling in mutants deficient in DNA topoisomerase I. J Bacteriol. 1993 Mar;175(6):1645–1655. doi: 10.1128/jb.175.6.1645-1655.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Olins P. O., Rangwala S. H. A novel sequence element derived from bacteriophage T7 mRNA acts as an enhancer of translation of the lacZ gene in Escherichia coli. J Biol Chem. 1989 Oct 15;264(29):16973–16976. [PubMed] [Google Scholar]
  15. Rogers E. J., Ambulos N. P., Jr, Lovett P. S. Ribosome hopping and translational frameshifting are inadequate alternatives to translational attenuation in cat-86 regulation. J Bacteriol. 1991 Dec;173(24):7881–7886. doi: 10.1128/jb.173.24.7881-7886.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Sandler S. J., Clark A. J. Factors affecting expression of the recF gene of Escherichia coli K-12. Gene. 1990 Jan 31;86(1):35–43. doi: 10.1016/0378-1119(90)90111-4. [DOI] [PubMed] [Google Scholar]
  17. Sandler S. J., Clark A. J. Use of high and low level overexpression plasmids to test mutant alleles of the recF gene of Escherichia coli K-12 for partial activity. Genetics. 1993 Nov;135(3):643–654. doi: 10.1093/genetics/135.3.643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Shapira S. K., Chou J., Richaud F. V., Casadaban M. J. New versatile plasmid vectors for expression of hybrid proteins coded by a cloned gene fused to lacZ gene sequences encoding an enzymatically active carboxy-terminal portion of beta-galactosidase. Gene. 1983 Nov;25(1):71–82. doi: 10.1016/0378-1119(83)90169-5. [DOI] [PubMed] [Google Scholar]
  19. Sprengart M. L., Fatscher H. P., Fuchs E. The initiation of translation in E. coli: apparent base pairing between the 16srRNA and downstream sequences of the mRNA. Nucleic Acids Res. 1990 Apr 11;18(7):1719–1723. doi: 10.1093/nar/18.7.1719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Sørensen M. A., Pedersen S. Absolute in vivo translation rates of individual codons in Escherichia coli. The two glutamic acid codons GAA and GAG are translated with a threefold difference in rate. J Mol Biol. 1991 Nov 20;222(2):265–280. doi: 10.1016/0022-2836(91)90211-n. [DOI] [PubMed] [Google Scholar]
  21. Thanaraj T. A., Pandit M. W. An additional ribosome-binding site on mRNA of highly expressed genes and a bifunctional site on the colicin fragment of 16S rRNA from Escherichia coli: important determinants of the efficiency of translation-initiation. Nucleic Acids Res. 1989 Apr 25;17(8):2973–2985. doi: 10.1093/nar/17.8.2973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Varenne S., Knibiehler M., Cavard D., Morlon J., Lazdunski C. Variable rate of polypeptide chain elongation for colicins A, E2 and E3. J Mol Biol. 1982 Jul 25;159(1):57–70. doi: 10.1016/0022-2836(82)90031-6. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Wolin S. L., Walter P. Ribosome pausing and stacking during translation of a eukaryotic mRNA. EMBO J. 1988 Nov;7(11):3559–3569. doi: 10.1002/j.1460-2075.1988.tb03233.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES