In vivo mitochondrial DNA-protein interactions in sea urchin eggs and embryos

Footprinting studies with the purine-modifying agent dimethyl sulphate were performed in Paracentrotus lividus eggs and embryos to analyze in vivo the interactions between protein and mitochondrial DNA. Footprinting in the sm all non-coding region and at the boundary between the ND5 and ND6 genes rev ealed two strong contact sites corresponding with the in vitro binding sequ ences of mitochondrial D-loop-Binding Protein (mtDBP). The analysis of the pause region of mtDNA replication showed a strong footprint corresponding w ith the binding site of the mitochondrial Pause region-Binding Protein-2 (m tPBP-2), but only a very weak signal at the binding site of the mitochondri al Pause region-Binding Protein-1 (mtPBP-1), which in vitro binds DNA with high efficiency. In vitro and in vivo analysis of the 3' end-region of the two rRNA genes showed no significant protein-DNA interactions, suggesting t hat, in contrast to mammals, the 3' ends of sea urchin mitochondrial rRNAs are not generated by a protein-dependent transcription termination event. T hese and other data support a model in which expression of mitochondrial ge nes in sea urchins is regulated post-transcriptionally. Footprinting at the five AT-rich consensus regions allowed the detection of a binding site in the non-coding region for an as-yet unidentified protein, mtAT-1BP. The occ upancy of this site appears to be developmentally regulated, being detectab le in the pluteus larval stage, but not in unfertilized eggs.