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. 1980 Jul;143(1):8–17. doi: 10.1128/jb.143.1.8-17.1980

Subunits of the Adenosine Triphosphatase Complex Translated In Vitro from the Escherichia coli unc Operon

J Allan Downie 1, Lyndall Langman 1, Graeme B Cox 1, Charles Yanofsky 1,†, Frank Gibson 1
PMCID: PMC294171  PMID: 6447144

Abstract

The unc operon of Escherichia coli was split into two fragments by the restriction endonuclease HindIII. The operator-proximal portion was cloned into plasmid pACYC184, forming plasmid pAN51, which included the genes uncB, uncE, and uncA. When plasmid pAN51 was used as template in an in vitro transcription/translation system, the α subunit (from the uncA gene) and δ subunit of the F1 adenosine triphosphatase (ATPase) were formed. In addition, three polypeptides of molecular weights 18,000, 17,000, and 14,000 were formed, and the significance of these polypeptides is discussed. The operator-distal portion of the unc operon was also cloned into plasmid pACYC184, forming plasmid pAN36, which included the uncD and uncC genes. When this plasmid was used as template in an in vitro transcription/translation system, the β subunit (from the uncD gene) and the ε subunit (from the uncC gene) of the F1 ATPase were formed. A polypeptide of a molecular weight similar to the ε subunit but of different net charge was also formed. Plasmid pAN45, carrying the complete unc operon, was isolated after digestion of a mixture of plasmids pAN51 and pAN36 with the restriction endonuclease HindIII and then religation with T4 deoxyribonucleic acid ligase. It was concluded that a HindIII restriction site occurred within the newly described uncG gene, which was shown, by complementation studies with Mu-induced mutants, to be located between the uncA and uncD genes to give the gene order uncBEAGDC. The uncG gene appears to code for the γ subunit of the F1 ATPase.

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Selected References

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  1. Bachmann B. J., Low K. B., Taylor A. L. Recalibrated linkage map of Escherichia coli K-12. Bacteriol Rev. 1976 Mar;40(1):116–167. doi: 10.1128/br.40.1.116-167.1976. [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. Barnes W. M. Plasmid detection and sizing in single colony lysates. Science. 1977 Jan 28;195(4276):393–394. doi: 10.1126/science.318764. [DOI] [PubMed] [Google Scholar]
  4. Cannon F. C., Dixon R. A., Postgate J. R. Derivation and properties of F-prime factors in Escherichia coli carrying nitrogen fixation genes from Klebsiella pneumoniae. J Gen Microbiol. 1976 Mar;93(1):111–125. doi: 10.1099/00221287-93-1-111. [DOI] [PubMed] [Google Scholar]
  5. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Clarke L., Carbon J. A colony bank containing synthetic Col El hybrid plasmids representative of the entire E. coli genome. Cell. 1976 Sep;9(1):91–99. doi: 10.1016/0092-8674(76)90055-6. [DOI] [PubMed] [Google Scholar]
  7. Cox G. B., Downie J. A., Fayle D. R., Gibson F., Radik J. Inhibition, by a protease inhibitor, of the solubilization of the F1-portion of the Mg2+-stimulated adenosine triphosphatase of Escherichia coli. J Bacteriol. 1978 Jan;133(1):287–292. doi: 10.1128/jb.133.1.287-292.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cox G. B., Downie J. A., Gibson F., Radik J. Genetic complementation between two mutant unc alleles (unc A401 and unc D409) affecting the Fl portion of the magnesium ion-stimulated adenosine triphosphatase of Escherichia coli K12. Biochem J. 1978 Mar 15;170(3):593–598. doi: 10.1042/bj1700593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cox G. B., Downie J. A. Isolation and characterization of mutants of Escherichia coli K-12 affected in oxidative phosphorylation of quinone biosynthesis. Methods Enzymol. 1979;56:106–117. doi: 10.1016/0076-6879(79)56013-3. [DOI] [PubMed] [Google Scholar]
  10. Cox G. B., Gibson F., McCann L. Reconstitution of oxidative phosphorylation and the adenosine triphosphate-dependent transhydrogenase activity by a combination of membrane fractions from unCA- and uncB- mutant strains of Escherichia coli K12. Biochem J. 1973 Aug;134(4):1015–1021. doi: 10.1042/bj1341015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Downie J. A., Gibson F., Cox G. B. Membrane adenosine triphosphatases of prokaryotic cells. Annu Rev Biochem. 1979;48:103–131. doi: 10.1146/annurev.bi.48.070179.000535. [DOI] [PubMed] [Google Scholar]
  12. Downie J. A., Senior A. E., Gibson F., Cox G. B. A fifth gene (uncE) in the operon concerned with oxidative phosphorylation in Escherichia coli. J Bacteriol. 1979 Feb;137(2):711–718. doi: 10.1128/jb.137.2.711-718.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dunn S. D. Identification of the altered subunit in the inactive F1ATPase of an Escherichia coli uncA mutant. Biochem Biophys Res Commun. 1978 May 30;82(2):596–602. doi: 10.1016/0006-291x(78)90916-6. [DOI] [PubMed] [Google Scholar]
  14. Fayle D. R., Downie J. A., Cox G. B., Gibson F., Radik J. Characterization of the mutant-unc D-gene product in a strain of Escherichia coli K12. An altered beta-subunit of the magnesium ion-stimulated adenosine triphosphatase. Biochem J. 1978 Jun 15;172(3):523–531. doi: 10.1042/bj1720523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Foster D. L., Fillingame R. H. Energy-transducing H+-ATPase of Escherichia coli. Purification, reconstitution, and subunit composition. J Biol Chem. 1979 Sep 10;254(17):8230–8236. [PubMed] [Google Scholar]
  16. Friedl P., Friedl C., Schairer H. U. The ATP synthetase of Escherichia coli K12: purification of the enzyme and reconstitution of energy-transducing activities. Eur J Biochem. 1979 Oct;100(1):175–180. doi: 10.1111/j.1432-1033.1979.tb02046.x. [DOI] [PubMed] [Google Scholar]
  17. Gibson F., Cox G. B., Downie J. A., Radik J. A mutation affecting a second component of the F0 portion of the magnesium ion-stimulated adenosine triphosphatase of Escherichia coli K12. The uncC424 allele. Biochem J. 1977 Apr 15;164(1):193–198. doi: 10.1042/bj1640193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gibson F., Cox G. B., Downie J. A., Radik J. Partial diploids of Escherichia coli carrying normal and mutant alleles affecting oxidative phosphorylation. Biochem J. 1977 Mar 15;162(3):665–670. doi: 10.1042/bj1620665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gibson F., Downie J. A., Cox G. B., Radik J. Mu-induced polarity in the unc operon of Escherichia coli. J Bacteriol. 1978 Jun;134(3):728–736. doi: 10.1128/jb.134.3.728-736.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hamer D. H., Thomas C. A., Jr Molecular cloning of DNA fragments produced by restriction endonucleases Sa1I and BamI. Proc Natl Acad Sci U S A. 1976 May;73(5):1537–1541. doi: 10.1073/pnas.73.5.1537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hare J. F. Purification and characterization of a dicyclohexylcarbodiimide-sensitive adenosine triphosphatase complex from membranes of Escherichia coli. Biochem Biophys Res Commun. 1975 Oct 27;66(4):1329–1337. doi: 10.1016/0006-291x(75)90505-7. [DOI] [PubMed] [Google Scholar]
  22. Kanazawa H., Saito S., Futai M. Coupling factor ATPase from Escherichia coli. An uncA mutant (uncA401) with defective alpha subunit. J Biochem. 1978 Dec;84(6):1513–1517. doi: 10.1093/oxfordjournals.jbchem.a132276. [DOI] [PubMed] [Google Scholar]
  23. Kanner B. I., Nelson N., Gutnick D. L. Differentiation between mutants of Escherichia coli K defective in oxidative phosphorylation. Biochim Biophys Acta. 1975 Sep 8;396(3):347–359. doi: 10.1016/0005-2728(75)90141-3. [DOI] [PubMed] [Google Scholar]
  24. Lederberg E. M., Cohen S. N. Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid. J Bacteriol. 1974 Sep;119(3):1072–1074. doi: 10.1128/jb.119.3.1072-1074.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Novick R. P., Clowes R. C., Cohen S. N., Curtiss R., 3rd, Datta N., Falkow S. Uniform nomenclature for bacterial plasmids: a proposal. Bacteriol Rev. 1976 Mar;40(1):168–189. doi: 10.1128/br.40.1.168-189.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  27. O'Farrell P. Z., Goodman H. M., O'Farrell P. H. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell. 1977 Dec;12(4):1133–1141. doi: 10.1016/0092-8674(77)90176-3. [DOI] [PubMed] [Google Scholar]
  28. Rood J. I., Laird A. J., Williams J. W. Cloning of the Escherichia coli K-12 dihydrofolate reductase gene following mu-mediated transposition. Gene. 1980 Feb;8(3):255–265. doi: 10.1016/0378-1119(80)90003-7. [DOI] [PubMed] [Google Scholar]
  29. Selker E., Brown K., Yanofsky C. Mitomycin C-induced expression of trpA of Salmonella typhimurium inserted into the plasmid ColE1. J Bacteriol. 1977 Jan;129(1):388–394. doi: 10.1128/jb.129.1.388-394.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Senior A. E., Downie J. A., Cox G. B., Gibson F., Langman L., Fayle D. R. The uncA gene codes for the alpha-subunit of the adenosine triphosphatase of Escherichia coli. Electrophoretic analysis of uncA mutant strains. Biochem J. 1979 Apr 15;180(1):103–109. doi: 10.1042/bj1800103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Senior A. E., Fayle D. R., Downie J. A., Gibson F., Cox G. B. Properties of membranes from mutant strains of Escherichia coli in which the beta-subunit of the adenosine triphosphatase is abnormal. Biochem J. 1979 Apr 15;180(1):111–118. doi: 10.1042/bj1800111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sone N., Yoshida M., Hirata H., Kagawa Y. Resolution of the membrane moiety of the H+-ATPase complex into two kinds of subunits. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4219–4223. doi: 10.1073/pnas.75.9.4219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Young I. G., Jaworowski A., Poulis M. I. Amplification of the respiratory NADH dehydrogenase of Escherichia coli by gene cloning. Gene. 1978 Sep;4(1):25–36. doi: 10.1016/0378-1119(78)90012-4. [DOI] [PubMed] [Google Scholar]
  34. Zalkin H., Yanofsky C., Squires C. L. Regulated in vitro synthesis of Escherichia coli tryptophan operon messenger ribonucleic acid and enzymes. J Biol Chem. 1974 Jan 25;249(2):465–475. [PubMed] [Google Scholar]

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