Characterization of primary transcripts and identification of transcription initiation sites on the heavy and light strands of mouse mitochondrial DNA

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Abstract
Total RNA from Ehrlich ascites mitochondria pretreated with RNase-free DNase was capped in vitro with [.alpha.-32P]GTP and guanylyl transferase. The capable RNAs representing the primary transcripts show a heterogeneous size distribution with four major species of 46, 63, 94, and 152 nucleotides and four minor species of 19, 24, 104, and 790 nucleotides in size. Hybridization with the D-loop DNA probes shows that the 19-nucleotide-long capped RNA is coded by the H-strand of mitochondrial DNA while the rest are coded by the L-strand. S1 nuclease mapping and primer extension analyses suggest the occurrence of a transcription initiation of H-strand at about 19 nucleotides upstream from the start of the tRNAPhe gene. All of the L-strand capable RNAs have a common 5'' end mapping to nucleotide 16,183 .+-. 5 of the genome. The 3'' ends of four major capable RNA species line up to the conserved sequences boxes, putative start sites of DH-DNA; and in fact about 2% of these capable species are found to exist as DNA-linked RNA under steady-state conditions. The 3'' end of the 790-nucleotide capable RNA lies close to the start of the tRNAPro gene, suggesting that it may be the true precursor of L-strand transcript endonucleolytically processed at the 3'' end. The level of L-strand-coded capable RNAs varies markedly under different growth conditions. Treatment with cycloheximide results in a reduction while chloramphenicol caused over 3-fold induction, suggesting that these "primer" RNAs may have an additional regulatory function.