Transcription of the HeLa Cell Mitochondrial Genome

The HeLa cell mitochondrial genome has been shown to encode, in addition to a number of tRNAs, two ribosomal RNAs and at least 17 discrete poly(A)-containing RNA species. A variety of techniques, including Southern blots, Northern blots and Berk and Sharp analyses, were utilized to orient the ribosomal and polyadenylated species with respect to a detailed restriction map. Thus, nineteen species were successfully localized to within an accuracy of about 30 to 40 nucleotides, thereby allowing a number of interesting observations to be made concerning the process of transcription of this genome. First, both the ribosomal RNAs and polyadenylated RNA components are transcribed colinearly; no intervening sequences exist. This is in dramatic contrast to the situation observed in mitochondrial DNA of some strains of yeast. Second, there exists no overlapping of the mature polyadenylated species, the ribosomal RNAs and the tRNAs (within the resolution described) and, with the exception of the D-loop region, the mitochondrial DNA sequences appear to be completely utilized for the synthesis of the transcripts. Third, a comparison of the positions of the DNA sequences which encode the polyadenylated transcripts with respect to those encoding the tRNAs shows that the majority of those transcripts are flanked at both 5'- and 3'-ends by a tRNA. Direct sequencing analyses have been used to investigate the precise relationship of the 5'- and 3'-ends of many of the polyadenylated transcripts (which are presumptive mRNAs) with respect to the ends of the tRNAs. Results obtained have demonstrated that those species which have been shown to be flanked by a tRNA gene at their 5'-end begin immediately following the tRNA 3'-terminal nucleotide. Correspondingly, the 3'-terminal nucleotide of seven of these RNA transcripts has been found to be immediately juxtaposed to the 5'-terminal nucleotide of the flanking tRNA. In one case, the 3'-terminal nucleotide is adjacent to the 5'-terminal nucleotide of the following mRNA. Moreover, an additional striking feature of these RNAs is that, with one possible exception, they either lack a 5'-end noncoding stretch, or contain an abbreviated version of it. Thus six species begin directly with AUG or A UA (which codes for methionine in human mitochondria), while three species contain those triplets from within one to eight nucleotides of the 5'-end. A model is proposed whereby structural features or sequences of tRNA specific transcripts play a role in the processing events which form the mature species. Specifically, the tRNA would provide a signal for a precise endonucleolytic cleavage event which would generate the mature species from a larger precursor. The results presented in this thesis, in correspondence with sequencing data (B. Barrell and F. Sanger, personal communication}, describe a transcriptional system characterized by simplicity, efficiency and economy.