Identification and in vitro capping of a primary transcript of human mitochondrial DNA.
A discrete primary transcript of HeLa mitochondrial DNA containing 12 S rRNA, tRNAPhe, and 19 +/- 2 nucleotides upstream has been identified. The 5' ends of heavy strand encoded RNAs adjacent to the D-loop region of mitochondrial DNA were mapped by primer extension and S1 nuclease protection to a site 19 +/- 2 nucleotides upstream from the start of tRNAPhe. Hybridization of a tRNAPhe-specific probe to a blot of mitochondrial RNA detected a discrete species with a molecular weight consistent with an RNA containing 12 S rRNA, tRNAPhe, and a short upstream leader sequence. The di- or triphosphate nature of the 5' end of this polycistronic RNA was demonstrated by in vitro capping with [alpha-32P]GTP and guanylyltransferase. S1 nuclease protection of the purified in vitro capped RNA established that the 5' end, 19 +/- 2 nucleotides upstream from tRNAPhe, is an initiation site for heavy strand transcription.
D001483:Base Sequence; D004262:DNA Restriction Enzymes; D004272:DNA, Mitochondrial / Q000235:genetics*; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D009693:Nucleic Acid Hybridization; D012315:RNA Caps / Q000235:genetics*; D012335:RNA, Ribosomal / Q000235:genetics; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics; D014158:Transcription, Genetic
D004272:DNA, Mitochondrial; D012315:RNA Caps; D012335:RNA, Ribosomal; D012346:RNA, Transfer, Amino Acyl; D004262:DNA Restriction Enzymes