Abstract
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(Leu)UUR genes, multiple mtTFA contacts were detected. Contact sites were located in the light and the heavy strand promoters and, in agreement with in vitro 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.
Highlights
The majority of mammalian mitochondrial1 DNA molecules possess a triple helix structure called D-loop due to the displacement of the parental H-strand by short nascent Hstrand chains
The termination of the H-strand ribosomal transcription unit depends on a mt termination factor, mTERF, that binds to a sequence located few bases downstream of the 5Ј end of the tRNALeuUUR gene [8], whereas the primary transcript processing seems to be mediated by a RNase P-like endonuclease, which should recognize the cloverleaf structure of the tRNA genes that separate most of the rRNA and mRNA genes [9]
Several reports have shown that isolated mitochondria from different mammalian sources are able to synthesize and process mt RNA in a way that closely resembles the in vivo process [21,22,23,24, 27,28,29]
Summary
The majority of mammalian mitochondrial (mt) DNA molecules possess a triple helix structure called D-loop due to the displacement of the parental H-strand by short nascent Hstrand chains. The termination of the H-strand ribosomal transcription unit depends on a mt termination factor, mTERF, that binds to a sequence located few bases downstream of the 5Ј end of the tRNALeuUUR gene [8], whereas the primary transcript processing seems to be mediated by a RNase P-like endonuclease, which should recognize the cloverleaf structure of the tRNA genes that separate most of the rRNA and mRNA genes [9]. We used an in vivo-like environment, made by rat liver isolated mitochondria, to investigate by means of in organello footprinting with dimethyl sulfate (DMS) and potassium permanganate (KMnO4) the occurrence of protein binding sites and of singlestranded DNA structures in the regulatory region and in the transcription termination region of rat mt DNA By these techniques we were able to detect, within the mitochondrion, multiple protein-DNA interactions and, for the first time, a singlestranded-DNA region located within CSB-1. The data obtained with this approach suggest that protein-induced mt DNA conformational changes play an important role in defining the 5Ј ends of the prominent H-DNA nascent chains
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