Identification of initiation sites for heavy-strand and light-strand transcription in human mitochondrial DNA.

Abstract

The initiation sites for heavy (H) and light (L) strand transcription in HeLa cell mitochondrial DNA have been investigated by mapping experiments utilizing in vitro "capped" mitochondrial RNA molecules or nascent RNA chains. Mitochondrial poly(A)-containing RNA molecules were labeled at their 5' ends with [a-32P]GTP and guanylyltransferase ("capping" enzyme) and mapped on the mitochondrial genome by DNA transfer hybridization and S1 nuclease protection experiments. A mapping site for the capped 5' ends was found on the H strand very near to the 5' terminus of the 12S rRNA gene, and another site was found on the L strand very near to the 5' terminus of the 7S RNA coding sequence. In parallel experiments, the 5' ends of the nascent chains isolated from mitochondrial DNA transcription complexes were similarly mapped very near to the 5' termini of the 12S rRNA gene and of the 7S RNA coding sequence. The in vitro capped RNA molecules and the nascent chains thus presumably identify the same transcriptional initiation sites on the H strand and the L strand. The occurrence of a second possible initiation site for H-strand transcription 90-110 nucleotides upstream of that described above-i.e., 20-40 nucleotides upstream of the tRNAPhe gene-had been previously indicated by a mapping analysis of the nascent RNA chains and has been confirmed in the present work. The presence of two initiation sites for H-strand transcription can be correlated with other types of evidence that point to two different transcription events leading to the synthesis of a polycistronic molecule corresponding to the almost entire H strand and to the synthesis of the rRNA species.Previous studies on mtDNA transcription in HeLa cells have shown that both strands are completely or almost completely transcribed (1,2). This conclusion has been recently confirmed for the heavy (H) strand by the observation that the discrete transcripts of this strand form a continuum extending over its entire length, with the exception of a relatively small region from coordinate 98/100 to coordinate 5/100 [relative to the origin ofreplication taken as 0/100 (3-5) (Fig. 1)]. In the present work, the initiation sites for heavy (H) and light (L) strand transcription in HeLa cell mtDNA have been investigated by an approach that has been used successfully for identifying the transcriptional initiation sites in yeast mtDNA (8,9). In particular, the 5' ends oftotal poly(A)-containing mitochondrial RNA possessing a di-or triphosphate, and therefore presumably resulting from transcriptional initiation, have been identified by taking advantage of the capacity of the guanylyltransferase ("capping" enzyme) to transfer GMP from GTP to di-or triphosphate-terminated polynucleotides (10- (6) The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisenent" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Effect of DNA conformation on the transcription of mitochondrial DNA

“…Selertivr franscription. Transcription was terminated prcmaturcly by the omission of GTP (1) or CTP (2). The DNA in its native form corresponds to the plasmids: a, pXlmBE containing a coding, non-promoter mlDNA fragment; b, pXlmH5 containing a noncoding region; c, pXlmE2 containing the promoter sequences; and d thc vcctor pUC18 and the ways that DNA-binding proteins could possibly affect transcription.…”

Mammalian transcription factor A is a core component of the mitochondrial transcription machinery

“…Transcription from LSP also produces the RNA primers required for initiation of mtDNA replication at the origin of the heavy strand (2)(3)(4). In vivo experiments have identified a second transcription initiation site (HSP2) downstream of HSP1, but the sequence requirements of this promoter remain to be defined (5,6).…”

Transcriptional requirements of the distal heavy-strand promoter of mtDNA

“…Intracellular levels of mature rRNAs are in great excess to the mRNAs (3). Although it was initially proposed that this imbalance was caused by the existence of a single initiation site, with most primary transcripts terminating before reaching the protein coding region, later studies used a combination of ribonucleotide incorporation assays and capping of precursor transcripts to suggest the existence of two distinct heavy-strand initiation sites in close proximity (4)(5)(6). The first heavy-strand promoter (HSP1), which lies fully within the Dloop, is responsible for the synthesis of a transcript composed of the 12S and 16S rRNAs and intervening tRNAs (Fig.…”