Altered translation of the uncC gene coding for the epsilon subunit of the F1F0-ATPase of Escherichia coli

Abstract

The nucleotide sequence of the previously described uncC424 allele was determined and found to be the same as that of a wild-type uncC gene. However, a G-A change occurred 7 nucleotides upstream from the translation start codon, changing the putative Shine-Dalgarno sequence from GAGG to GAAG. Four revertant strains were examined. In one revertant, which had normal growth and membrane properties, a single base deletion had occurred to re-form the Shine-Dalgarno sequence GAGG 1 nucleotide closer to the translation start codon. A second revertant had a single base deletion in the preceding uncD gene, causing an extension of the ,B subunit by 6 amino acids and an increase, presumably by translational coupling, in the amount of e subunit. The third and fourth revertant strains were phenotypically similar and had either C-*T or G-*T changes 18 or 19 nucleotides, respectively, upstream from the translation start codon.The F1FO-ATPase complex of mitochondrial, chloroplast, and bacterial membranes catalyses the terminal reaction in oxidative phosphorylation. This highly conserved complex can be dissociated into a water-soluble portion (F1-ATPase), which catalyzes the hydrolysis of ATP, and an intrinsic membrane portion (FO), which forms a proton pore (23). The genes coding for the subunits of the F1F0-ATPase complex in Escherichia coli make up the unc operon at about 83.5 min on the chromosome, and the complete DNA sequence has been determined (26). The unc operon consists of nine genes in the order IBEFHAGDC corresponding to the subunits i, a, c, b, 5, a, ay, 1, and e. The uncl gene product is thought (12), using site-directed mutagenesis, obtained evidence suggesting that secondary structure in the mRNA and the use of a less efficient initiation codon play a part in restricting translation initiation of the uncF mRNA segment.In this paper we characterize a mutant allele (uncC424) (7, 9) which affects the amount of the e subunit formed and four revertant strains which, by various means, have overcome the defect. MATERIALS AND METHODS Enzymes and chemicals. Restriction endonucleases and T4

An upstream uncD sequence modulates translation of Escherichia coli uncC

“…2) are consistent with the formation of this structure blocking initiation of a synthesis. Furthermore, Cox and co-woikers (3) have found that mutations of either the G or the C indicated (Fig. 4, asterisk) modulates E expression, rather than the translation of that RNA.…”