IDENTIFICATION BY IN ORGANELLO FOOTPRINTING OF PROTEIN CONTACT SITES AND OF SINGLE-STRANDED-DNA SEQUENCES IN THE REGULATORY REGION OF RAT MITOCHONDRIAL-DNA - PROTEIN-BINDING SITES AND SINGLE-STRANDED-DNA REGIONS IN ISOLATED RAT-LIVER MITOCHONDRIA

Footprinting studies with the purine-modifying reagent dimethyl sulfate and with the single-stranded DNA probing reagent potassium permanganate were carried out in isolated mitochondria from rat liver. Dimethyl sulfate footprinting allowed the detection of protein-DNA interactions within the rat analogues of the human binding sites for the transcription termination factor mTERF and for the transcription activating factor mt-TFA. Although mTERF contacts were localized only at the boundary between the 16S rRNA/tRNA(UUR)(Leu) genes, multiple mtTFA contacts were detected, Contact sites were located in the light and the heavy strand promoters and, in agreement with in nitro footprinting data on human mitochondria, between the conserved sequence blocks (CSB) 1 and 2 and inside CSB-1. Potassium permanganate footprinting allowed detection of a 25-base pair region entirely contained in CSB-1 in which both strands were permanganate-reactive. No permanganate reactivity was associated with the other regions of the D-loop, including CSB-2 and -3, and with the mTERF contact site. We hypothesize that the single-stranded DNA at CSB-1 may be due to a profound helix distortion induced by mtTFA binding or be associated with a RNA polymerase pause site. In any case the location in CSB-1 of the 3' end of the most abundant replication primer and of the 5' end of the prominent D-loop DNA suggests that protein-induced DNA conformational changes play an important role in directing the transition from transcription to replication in mammalian mitochondria.