In vitro synthesis of the F0 and F1 components of the proton translocating ATPase of Escherichia coli.

W.S Brusilow,R.P Gunsalus,E.C Hardeman,K.P Decker,R.D Simoni

doi:10.1016/s0021-9258(19)69576-1

Abstract

Specialized lambda transducing phage DNA containing the unc region of the Escherichia coli chromosome was used as template to direct an in vitro transcription/translation system. The results demonstrated synthesis of seven of the eight polypeptides of the proton translocating ATPase of this organism. The three polypeptides a, b, and c, constituting the F0 portion of the complex, were resolved by sodium dodecyl sulfatepolyacrylamide gel analysis and have apparent molecular weights (Mr = 24,000, 18,000, and 8,000-9,000) similar to the corresponding proteins produced in vivo. In addition, the alpha, beta, delta, and epsilon polypeptides of the F1 portion of the ATPase were also detected and their molecular weights correspond to the in vivo peptides. A 4.3-kilobase HindIII-generated lambda unc DNA fragment was cloned onto plasmid vectors and was demonstrated to contain the genes for the three F0 and two of the F1 (alpha, delta) subunits. In addition, the polypeptides synthesized in vitro were precipitable with antibody prepared against purified F1.

Highlights

The unc region from the E. coli chromosome onto plasmid vectors
Specialized X transducing phageDNA containing the sized and are presumably related to the polypeptidofesthe Fo unc region oftheEscherichiacolichromosome was portion of the complex. It was noted by the authorsh,owever, used as template to direct an in vitm transcription/ that thesein vitro polypeptidesdid not correspond in apparent translation system
The results demonstrated synthesis molecular weights to theknown in vivo polypeptides of the Fo of seven of the eight polypeptides of the proton trans- portion as determined independentlyby Foster andFillingame locating ATPase of this organism

Summary

Introduction

The unc region from the E. coli chromosome onto plasmid vectors. They reported thine vitro synthesisof all the peptides (a,p, y, 6, e ) of the F1portion of the ATPasecomplex using an. The three polypeptides a, b,and c, constituting the Fo portionofthe complex,were resolved bysodiumdodecyl sulfatepolyacrylamide gel analysis and have apparent molecular weights (Mr= 24,000, 18,000, and 8,000-9,000) similar to the corresponding proteins produced invivo.

Results

Conclusion