IDENTIFICATION OF A MITOCHONDRIAL RNA-POLYMERASE IN THE CRUSTACEAN ARTEMIA-FRANCISCANA

Mitochondrial RNA polymerase activity has been isolated from the crust acean Artemia franciscana at two stages of development, dormant embryo and developing larva. The preparations were obtained from purified mi tochondria and the polymerase activity was purified by heparin-Sepharo se chromatography. The presumed polymerase has a molecular mass of abo ut 120 kDa and a 7.4S sedimentation coefficient. The biochemical chara cterization of the enzymatic reaction identified our RNA polymerase pr eparations as mitochondrial. The transcription initiation sites of Art emia mtDNA were characterized recently in our laboratory (J. A Carrode guas and C. G. Vallejo, fur. J. Biochem. 250, 514-523, 1997). Artemia mtDNA fragments comprising the transcription initiation sites were tra nscribed by the partially purified polymerase preparation from the two developmental stages, but the transcription turned out to be unspecif ic. DNAse I footprinting analysis of a main transcription initiation s ite-containing DNA fragment revealed a protected region around the ini tiation site fl position, when using a crude polymerase preparation. H owever, the protected region was not observed with the purified prepar ation. The results altogether suggest that a specificity factor is los t during purification. Based on the footprinting data, we suggest that the sequence from positions -6 to +13 of the main transcription initi ation site in the Artemia mitochondrial DNA is the binding site of the homologous RNA polymerase holoenzyme. (C) 1998 Academic Press.