Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1989 Jun;171(6):3039–3045. doi: 10.1128/jb.171.6.3039-3045.1989

Use of lacZ fusions to measure in vivo expression of the first three genes of the Escherichia coli unc operon.

K A Solomon 1, D K Hsu 1, W S Brusilow 1
PMCID: PMC210012  PMID: 2524469

Abstract

We have constructed in-frame lacZ protein fusions to the first three genes of the Escherichia coli unc operon, which codes for the subunits of the proton-translocating ATPase. We have used these constructions to measure the relative in vivo expression of these genes. The second and third genes, uncB and uncE, which code for the a and c subunits of the F0 sector, were expressed at relative levels of approximately 1:10, although the measured expression of uncB depended upon how much of the gene was fused to lacZ. These rates compared favorably with the relative numbers of a and c subunits (a1:c10) in the purified F1F0 complex. The in vivo expression of uncI, the first gene of the operon, was very low, at best 10 to 20 times less than the expression of uncB.

Full text

PDF
3039

Images in this article

3043Image
on p.3043

Selected References

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

  1. Angov E., Brusilow W. S. Use of lac fusions to measure in vivo regulation of expression of Escherichia coli proton-translocating ATPase (unc) genes. J Bacteriol. 1988 Jan;170(1):459–462. doi: 10.1128/jb.170.1.459-462.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Bremer E., Silhavy T. J., Weisemann J. M., Weinstock G. M. Lambda placMu: a transposable derivative of bacteriophage lambda for creating lacZ protein fusions in a single step. J Bacteriol. 1984 Jun;158(3):1084–1093. doi: 10.1128/jb.158.3.1084-1093.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brusilow W. S., Klionsky D. J., Simoni R. D. Differential polypeptide synthesis of the proton-translocating ATPase of Escherichia coli. J Bacteriol. 1982 Sep;151(3):1363–1371. doi: 10.1128/jb.151.3.1363-1371.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brusilow W. S., Porter A. C., Simoni R. D. Cloning and expression of uncI, the first gene of the unc operon of Escherichia coli. J Bacteriol. 1983 Sep;155(3):1265–1270. doi: 10.1128/jb.155.3.1265-1270.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
  7. Foster D. L., Fillingame R. H. Stoichiometry of subunits in the H+-ATPase complex of Escherichia coli. J Biol Chem. 1982 Feb 25;257(4):2009–2015. [PubMed] [Google Scholar]
  8. Fowler A. V., Zabin I. Purification, structure, and properties of hybrid beta-galactosidase proteins. J Biol Chem. 1983 Dec 10;258(23):14354–14358. [PubMed] [Google Scholar]
  9. Froshauer S., Green G. N., Boyd D., McGovern K., Beckwith J. Genetic analysis of the membrane insertion and topology of MalF, a cytoplasmic membrane protein of Escherichia coli. J Mol Biol. 1988 Apr 5;200(3):501–511. doi: 10.1016/0022-2836(88)90539-6. [DOI] [PubMed] [Google Scholar]
  10. Futai M., Kanazawa H. Structure and function of proton-translocating adenosine triphosphatase (F0F1): biochemical and molecular biological approaches. Microbiol Rev. 1983 Sep;47(3):285–312. doi: 10.1128/mr.47.3.285-312.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gay N. J. Construction and characterization of an Escherichia coli strain with a uncI mutation. J Bacteriol. 1984 Jun;158(3):820–825. doi: 10.1128/jb.158.3.820-825.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Grove C. L., Gunsalus R. P. Regulation of the aroH operon of Escherichia coli by the tryptophan repressor. J Bacteriol. 1987 May;169(5):2158–2164. doi: 10.1128/jb.169.5.2158-2164.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gunsalus R. P., Brusilow W. S., Simoni R. D. Gene order and gene-polypeptide relationships of the proton-translocating ATPase operon (unc) of Escherichia coli. Proc Natl Acad Sci U S A. 1982 Jan;79(2):320–324. doi: 10.1073/pnas.79.2.320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jones H. M., Brajkovich C. M., Gunsalus R. P. In vivo 5' terminus and length of the mRNA for the proton-translocating ATPase (unc) operon of Escherichia coli. J Bacteriol. 1983 Sep;155(3):1279–1287. doi: 10.1128/jb.155.3.1279-1287.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. McCarthy J. E., Schairer H. U., Sebald W. Translational initiation frequency of atp genes from Escherichia coli: identification of an intercistronic sequence that enhances translation. EMBO J. 1985 Feb;4(2):519–526. doi: 10.1002/j.1460-2075.1985.tb03659.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. McCarthy J. E., Sebald W., Gross G., Lammers R. Enhancement of translational efficiency by the Escherichia coli atpE translational initiation region: its fusion with two human genes. Gene. 1986;41(2-3):201–206. doi: 10.1016/0378-1119(86)90099-5. [DOI] [PubMed] [Google Scholar]
  17. Nielsen J., Hansen F. G., Hoppe J., Friedl P., von Meyenburg K. The nucleotide sequence of the atp genes coding for the F0 subunits a, b, c and the F1 subunit delta of the membrane bound ATP synthase of Escherichia coli. Mol Gen Genet. 1981;184(1):33–39. doi: 10.1007/BF00271191. [DOI] [PubMed] [Google Scholar]
  18. Ostrow K. S., Silhavy T. J., Garrett S. cis-acting sites required for osmoregulation of ompF expression in Escherichia coli K-12. J Bacteriol. 1986 Dec;168(3):1165–1171. doi: 10.1128/jb.168.3.1165-1171.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Packman S., Sly W. S. Constitutive lambda DNA replication by lambda-C17, a regulatory mutant related to virulence. Virology. 1968 Apr;34(4):778–789. doi: 10.1016/0042-6822(68)90099-8. [DOI] [PubMed] [Google Scholar]
  20. Schneider E., Altendorf K. Bacterial adenosine 5'-triphosphate synthase (F1F0): purification and reconstitution of F0 complexes and biochemical and functional characterization of their subunits. Microbiol Rev. 1987 Dec;51(4):477–497. doi: 10.1128/mr.51.4.477-497.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Senior A. E., Wise J. G. The proton-ATPase of bacteria and mitochondria. J Membr Biol. 1983;73(2):105–124. doi: 10.1007/BF01870434. [DOI] [PubMed] [Google Scholar]
  22. Silhavy T. J., Beckwith J. R. Uses of lac fusions for the study of biological problems. Microbiol Rev. 1985 Dec;49(4):398–418. doi: 10.1128/mr.49.4.398-418.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sly W. S., Rabideau K. Mechanism of lambda-c17cI virulence. J Mol Biol. 1969 Jun 28;42(3):385–400. doi: 10.1016/0022-2836(69)90231-9. [DOI] [PubMed] [Google Scholar]
  24. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  25. Vik S. B., Cain B. D., Chun K. T., Simoni R. D. Mutagenesis of the alpha subunit of the F1Fo-ATPase from Escherichia coli. Mutations at Glu-196, Pro-190, and Ser-199. J Biol Chem. 1988 May 15;263(14):6599–6605. [PubMed] [Google Scholar]
  26. von Meyenburg K., Jørgensen B. B., Nielsen J., Hansen F. G. Promoters of the atp operon coding for the membrane-bound ATP synthase of Escherichia coli mapped by Tn10 insertion mutations. Mol Gen Genet. 1982;188(2):240–248. doi: 10.1007/BF00332682. [DOI] [PubMed] [Google Scholar]

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

RESOURCES