Transcriptional requirements of the distal heavy-strand promoter of mtDNA

Abstract

The heavy strand of mtDNA contains two promoters with nonoverlapping functions. The role of the minor heavy-strand promoter (HSP2) is controversial, because the promoter has been difficult to activate in an in vitro system. We have isolated HSP2 by excluding its interaction with the more powerful HSP1 promoter, and we find that it is transcribed efficiently by recombinant mtRNA polymerase and mitochondrial transcription factor B2. The mitochondrial transcription factor A is not required for initiation, but it has the ability to alternatively activate and repress the HSP2 transcriptional unit depending on the ratio between mitochondrial transcription factor A and other transcription factors. The positioning of transcriptional initiation agrees with our current understanding of HSP2 activity in vivo. Serial deletion of HSP2 shows that only proximal sequences are required. Several mutations, including the disruption of a polycytosine track upstream of the HSP2 initiation site, influence transcriptional activity. Transcription from HSP2 is also observed when HeLa cell mitochondrial extract is used as the source of mitochondrial polymerase, and this transcription is maintained when HSP2 is provided in proper spacing and context to the HSP1 promoter. Studies of the linked heavy-strand promoters show that they are differentially regulated by ATP dosage. We conclude that HSP2 is transcribed and has features that allow it to regulate mitochondrial mRNA synthesis.mitochondrial biology | organelle T he mitochondrial genome (mtDNA) encodes 13 proteins that are constituents of the electron transport chain and ATP synthase. Transcription of mtDNA proceeds bidirectionally, radiating out from the noncoding D-loop. The light-strand promoter (LSP) is required for the synthesis of ND6 mRNA and eight of the mitochondrial tRNAs (1). Transcription from LSP also primes the origin of heavy-strand replication, which is the first step in the synthesis of mtDNA (2).The heavy strand encodes 12 mRNAs, both mitochondrial rRNAs and the remaining tRNAs. 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 (4-6). 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. 1A). The minor or distal heavy-strand promoter (HSP2) initiates within the sequence of the mitochondrial tRNA for phenylalanine. HSP2-initiated transcription produces the remaining mitochondrial mRNAs by processing nearly the full length of the mtDNA.Studies of HSP2 promoter function are complicated by its unusual position, both within a tRNA-...

Transcription from the second heavy-strand promoter of human mtDNA is repressed by transcription factor A in vitro

“…S2). Our observations are consistent with the S1 mapping and 5′ RACE experiments of HSP2 in vitro transcripts in the work by Zollo et al (15). However, these start sites do not agree with the TSS at position 646 inferred from S1 mapping of HSP2 transcripts isolated from cells (12).…”

“…We conclude that more than one TSS exists for HSP2 in vitro; one TSS is at position 644. The work by Zollo et al (15) reached the same conclusion.…”

Mitochondrial transcription in mammalian cells

“…Importantly, in vitro , MTERF1 stimulates HSP1, but not HSP2 activity (21), further supporting the existence of two independent H-strand promoters. The activity of both HSP promoters can also be observed in an in vitro system, although in this system the major transcription start site of HSP2 maps to A644 instead C646 (22, 23). While the in vivo and in vitro transcription start sites of HSP2 are in reasonable agreement, this discrepancy raises an interesting possibility of regulation of the choice of HSP2 transcription start site by extrinsic factors like template topology, additional transacting factors, and/or nucleotide pools (22).…”

“…However, specific transcription initiation at HSP1, HSP2 and LSP promoters has been demonstrated in vitro in the absence of TFAM (22, 23, 26–29), which led to a two-component model in which TFAM is ascribed the role of transcriptional activator instead of being an obligate core component of the transcription initiation complex (27). This model, nevertheless, was challenged on the grounds that the experimental conditions used in those assays may not faithfully recapitulate those found in vivo because they favor promoter “breathing” (i.e.…”

“…Low-level transcription from all three mitochondrial promoters can be observed in vitro without TFAM (22, 23, 26–29). Varying TFAM concentrations in transcription reactions at all three mitochondrial promoters in vitro has a biphasic effect: both stimulation and repression were observed at different concentrations, but the principal effect at higher concentrations was to suppress transcription (26, 66).…”