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 sulfat e and with the single-stranded DNA probing reagent potassium permangan ate were carried out in isolated mitochondria from rat liver. Dimethyl sulfate footprinting allowed the detection of protein-DNA interaction s within the rat analogues of the human binding sites for the transcri ption termination factor mTERF and for the transcription activating fa ctor mt-TFA. Although mTERF contacts were localized only at the bounda ry between the 16S rRNA/tRNA(UUR)(Leu) genes, multiple mtTFA contacts were detected, Contact sites were located in the light and the heavy s trand promoters and, in agreement with in nitro footprinting data on h uman mitochondria, between the conserved sequence blocks (CSB) 1 and 2 and inside CSB-1. Potassium permanganate footprinting allowed detecti on of a 25-base pair region entirely contained in CSB-1 in which both strands were permanganate-reactive. No permanganate reactivity was ass ociated with the other regions of the D-loop, including CSB-2 and -3, and with the mTERF contact site. We hypothesize that the single-strand ed DNA at CSB-1 may be due to a profound helix distortion induced by m tTFA 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 replica tion primer and of the 5' end of the prominent D-loop DNA suggests tha t protein-induced DNA conformational changes play an important role in directing the transition from transcription to replication in mammali an mitochondria.