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

Abstract

The unc operon of Escherichia coli was split into two fragments by the restriction endonuclease Hin dIII. 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 F 1 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 F 1 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 Hin dIII and then religation with T4 deoxyribonucleic acid ligase. It was concluded that a Hin dIII 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 F 1 ATPase.

An additional acidic residue in the membrane portion of the b-subunit of the energy-transducing adenosine triphosphatase of Escherichia coli affects both assembly and function

“…Pvull Hinfl Rsal Alul (uncB+E+F+H+A+) (Downie et al, 1980). One such colony was purified (strain AN 1977) and the plasmid carried by this strain was designated pAN181.…”

“…The plasmid pAN181 was mixed with plasmid pAN36 (uncD+ C+) (Downie et al, 1980), and the mixture digested with the restriction endonuclease HindIII. The mixture was then treated with virus-T4 DNA ligase and used to transform strain AN 1570 (uncG428, recA) selecting for growth on succinate/chloramphenicol-containing medium.…”

“…The techniques used for genetic experiments were as outlined previously (Gibson et al, 1977bDownie et al, 1980). Prepart7tion ofplasmid and chromosomal DNA Plasmid DNA was prepared as described by Selker et al (1977).…”

“…Notes and references Partial diploid strain carrying uncF476 allele on both Fplasmid and chromosome. Isolated as described previously (Gibson et al, 1977b) Strain carrying uncB402 allele in AN346 recA background (Butlin et al, 1973;Gibson et al, 1977b) The present paper Strain carrying uncF476 allele in AN346 recA background The present paper Prepared by transduction of uncG428 allele into an ilvC derivative of C600 (Downie et al, 1980 The present paper Revertant of AN 1515 (the present paper) Partial revertant of AN1515 (the present paper) Strain carrying uncF476 allele in AN346 background ) Partial-diploid strain carrying unc alleles from AN 1953 on an F-plasmid, constructed as described previously (Gibson et al, 1977b) recA derivative of AN 1953 Obtained by mating AN780 with AN2033 (the present paper) Obtained by mating AN780 with AN 1521 ) (the present paper) Obtained by mating AN819 with AN2023 (the present paper) Obtained by mating AN819 with AN 1521 Downie et al (1980) The present paper F-plasmid carrying uncF476 allele isolated as described previously (Gibson et al, 1977b) The present paper F-plasmid carrying the mutant unc alleles from AN 1953 isolated as described previously (Gibson et al, 1977b) F-plasmid carrying uncA401 allele F-plasmid carrying uncD409 allele 1984 44 Escherichia coli uncF mutants DNA sequencing Nucleotide sequences were determined by the method of Maxam & Gilbert (1980). End-labelling of DNA was carried out by using DNA polymerase and deoxyadenosine [a-32P]triphosphate (Maxam & Gilbert, 1980).…”

“…The protein coded for by the uncI gene does not appear to be a structural component of the F1FO-ATPase complex (Gay & Walker, 1981). The uncBEF genes code for the a-, c-and bsubunits respectively of the membrane sector of the ATPase , whereas the uncHAGDC genes code for the b-, a-, y-, fand ssubunits respectively of the F1-ATPase (Downie et al, 1980;Gay & Walker, 1981;Gunsalus et al, 1982).…”