Studies of 8-azido-ATP adducts reveal two mechanisms by which ATP binding to cytochrome c could inhibit respiration

We have proposed that the binding of ATP at a site of substantial affinity and specificity could regulate the activity of cytochrome c with its physiological partners and thus the overall efficiency of mitochondrial electron transport. We now describe the use of ATP affinity-labeled protein to test the effect of occupancy of that site, which includes the invariant arginine 91, on the activity of cytochrome c with purified cytochrome c reductase and oxidase and its association with the mitochondrial inner membrane. Electron-transfer activities with the reductase and oxidase were inhibited by site occupancy to 41% and 11-15% of native values, respectively. The marked difference in the degree of inhibition of activity that distinguishes the reactions with the two major physiological partners was sufficient to cause, in whole mitochondria, a demonstrable shift from a situation in which there is a rate-limiting transfer from the reductase to cytochrome c, to a state where rates are more evenly matched for transfers between cytochrome c and the two redox partners. Site occupancy also substantially reduces the ionic strength necessary for half-maximal dissociation of cytochrome c from the membrane. These data imply that the decreased efficiency of electron transfer caused by ATP attachment can be attributed to a decrease in the protein's activity with individual physiological partners, possibly compounded with a decrease in its affinity for the inner mitochondrial membrane, and suggest that feedback regulation by ATP of cellular respiration operates in like manner.     

Sequence-specific single-strand RNA binding protein encoded by the human LINE-1 retrotransposon

Previous experiments using human teratocarcinoma cells indicated that p40, the protein encoded by the first open reading frame (ORF) of the human LINE-1 (L1Hs) retrotransposon, occurs in a large cytoplasmic ribonucleoprotein complex in direct association with L1Hs RNA(s), the p40 RNP complex. We have now investigated the interaction between partially purified p40 and L1Hs RNA in vitro using an RNA binding assay dependent on co-immunoprecipitation of p40 and bound RNA. These experiments identified two p40 binding sites on the full-length sense strand L1Hs RNA. Both sites are in the second ORF of the 6000 nt RNA: site A between residues 1999 and 2039 and site B between residues 4839 and 4875. The two RNA segments share homologous regions. Experiments involving UV cross-linking followed by immunoprecipitation indicate that p40 in the in vitro complex is directly associated with L1Hs RNA, as it is in the p40 RNP complex found in teratocarcinoma cells. Binding and competition experiments demonstrate that p40 binds to single-stranded RNA containing a p40 binding site, but not to single-stranded or double-stranded DNA, double-stranded RNA or a DNA-RNA hybrid containing a binding site sequence. Thus, p40 appears to be a sequence-specific, single-strand RNA binding protein.