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Transcriptional Requirements of the Distal Heavy-Strand Promoter of Mtdna

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Transcriptional Requirements of the Distal Heavy-Strand Promoter of Mtdna Transcriptional requirements of the distal heavy-strand promoter of mtDNA Ornella Zolloa, Valeria Tirantib, and Neal Sondheimera,c,1 aDivision of Child Rehabilitation, The Children’s Hospital of Philadelphia and cDepartment of Pediatrics, University of Pennsylvania, Philadelphia, PA 19104; and bDivision of Molecular Neurogenetics, Foundation Neurological Institute “C. Besta”, 20126 Milan, Italy Edited* by Douglas C. Wallace, Center for Mitochondrial and Epigenomic Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA, and approved March 6, 2012 (received for review November 12, 2011) The heavy strand of mtDNA contains two promoters with non- HSP2 in cells was precisely mapped to C646 using primer ex- overlapping functions. The role of the minor heavy-strand pro- tension and S1 digest of nascent transcripts from HeLa cells (7). moter (HSP2) is controversial, because the promoter has been However, recent studies that examined the minimal set of pro- difficult to activate in an in vitro system. We have isolated HSP2 by teins required for mitochondrial transcription in vitro were un- excluding its interaction with the more powerful HSP1 promoter, able to identify any transcription arising from HSP2 (8, 9). To and we find that it is transcribed efficiently by recombinant resolve this apparent conflict, we investigated transcription from mtRNA polymerase and mitochondrial transcription factor B2. The HSP2 in vitro using recombinant proteins and HeLa cell mito- mitochondrial transcription factor A is not required for initiation, chondrial extracts. We find that the promoter is active, responsive but it has the ability to alternatively activate and repress the HSP2 to mitochondrial transcription factor A (TFAM) dosage, and transcriptional unit depending on the ratio between mitochon- sensitive to mutations in upstream sequence. drial transcription factor A and other transcription factors. The positioning of transcriptional initiation agrees with our current Results understanding of HSP2 activity in vivo. Serial deletion of HSP2 Requirements for HSP2 Transcription. The mitochondrial heavy- shows that only proximal sequences are required. Several muta- strand promoters are closely spaced, and the isolation of HSP2 tions, including the disruption of a polycytosine track upstream of was required for visualization of its activity. When templates the HSP2 initiation site, influence transcriptional activity. Tran- containing both the HSP1 and HSP2 promoters (spanning BIOCHEMISTRY scription from HSP2 is also observed when HeLa cell mitochondrial nucleotides 460–801) were transcribed by recombinant mtRNA extract is used as the source of mitochondrial polymerase, and polymerase (POLRMT), TFAM, and mitochondrial transcrip- this transcription is maintained when HSP2 is provided in proper tion factor B2 (TFB2M), we observed products corresponding to spacing and context to the HSP1 promoter. Studies of the linked runoff transcription from HSP1 alone (Fig. 1B). After truncating heavy-strand promoters show that they are differentially regu- the promoter so that it lacked HSP1 (nucleotides 583–801), we lated by ATP dosage. We conclude that HSP2 is transcribed and observed species corresponding to runoff transcription from the has features that allow it to regulate mitochondrial mRNA predicted HSP2 promoter at nucleotide 646. The difference synthesis. between the relative activity of the two promoters was striking— HSP1 was greater than 100-fold more highly expressed under mitochondrial biology | organelle identical conditions. Next, we evaluated different combinations of recombinant he mitochondrial genome (mtDNA) encodes 13 proteins that proteins to determine which were required for HSP2 activity. Tare constituents of the electron transport chain and ATP Although a requirement for TFAM in mitochondrial transcrip- synthase. Transcription of mtDNA proceeds bidirectionally, ra- tion was previously assumed, it has been suggested more recently diating out from the noncoding D-loop. The light-strand pro- that TFAM is dispensable for HSP1 transcription by POLRMT moter (LSP) is required for the synthesis of ND6 mRNA and and TFB2M (9). Template-dependent transcriptional activity eight of the mitochondrial tRNAs (1). Transcription from LSP was seen when the three proteins were combined in equimolar also primes the origin of heavy-strand replication, which is the ratio but was equally robust in reactions where TFAM was ex- C first step in the synthesis of mtDNA (2). cluded (Fig. 1 ). No transcription was observed when either The heavy strand encodes 12 mRNAs, both mitochondrial POLRMT or TFB2M was excluded. rRNAs and the remaining tRNAs. Intracellular levels of mature To determine the span of the functional HSP2 promoter, we created templates with truncations upstream of the site of initi- rRNAs are in great excess to the mRNAs (3). Although it was – initially proposed that this imbalance was caused by the existence ation. A construct (nucleotides 623 801) extending 20 nt up- of a single initiation site, with most primary transcripts termi- stream had greatly reduced activity compared with the larger nucleotides 583–801 template. As expected, a deletion that re- nating before reaching the protein coding region, later studies moved the predicted start site (nucleotides 663–801) was inactive used a combination of ribonucleotide incorporation assays and (Fig. 1D). This finding suggests that the promoter is enhanced by capping of precursor transcripts to suggest the existence of two – sequence at some distance from the start site but that only a distinct heavy-strand initiation sites in close proximity (4 6). The ∼ fi short sequence spanning 20 nt upstream is absolutely required rst heavy-strand promoter (HSP1), which lies fully within the D- for initiation. loop, 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 Author contributions: N.S. designed research; O.Z. and N.S. performed research; V.T. the sequence of the mitochondrial tRNA for phenylalanine. contributed new reagents/analytic tools; O.Z. and N.S. analyzed data; and O.Z. and N.S. HSP2-initiated transcription produces the remaining mitochon- wrote the paper. drial mRNAs by processing nearly the full length of the mtDNA. The authors declare no conflict of interest. Studies of HSP2 promoter function are complicated by its *This Direct Submission article had a prearranged editor. unusual position, both within a tRNA-coding gene sequence 1To whom correspondence should be addressed. E-mail: [email protected]. fi ′ and with its rst transcribed base in close proximity to the 5 end This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. of the 12S rRNA. The 5′ end of the transcript generated from 1073/pnas.1118594109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1118594109 PNAS Early Edition | 1of5 Downloaded by guest on September 29, 2021 HSP1 HSP2 template (100nM) - + + + + A C POLRMT (100nM) + - + + + TrnF 12S TFB2M (100nM) + + - + + 460 561 577 647 801 583-801 TFAM (100nM) + + + - + 460-801 175 623-801 663-801 B 150 460-801 1% 5% no DNA 583-801 25bp ladder 25% 10% 50% 1 108- * 10 AN D 08-385 300 8 - D 366 326 250 on HSP1 ladder 200 * 175 175 HSP2 150 150 125 125 Fig. 1. In vitro transcription of HSP2 using purified transcription factors. (A) HSP2 and constructs used in this study. (B) Transcription of a 100-nM template containing HSP2 alone (nucleotides 583–801) by recombinant POLRMT, TFAM, and TFB2M (100 nM each) generated a runoff product of the expected size. Transcription using an equimolar template containing both promoters (nucleotides 460–801) produced strong products from HSP1 transcription but no detectible species from HSP2. To facilitate comparison between the promoters, the products of the HSP1-driven transcription were diluted before loading as shown. *RNA matching the size of input DNA is typically produced by POLRMT exposed to linear templates because of a nonspecific opening and tran- scription from DNA ends. (C) Combinations of recombinant transcription factors at 100 nM were used to transcribe HSP2. (D) In vitro transcription assays were performed using several truncations of the predicted HSP2 promoter. Confirmation of the Site of Initiation by S1 Analysis. An alignment of transcription reactions driven by either recombinant proteins or the initiation sites for the mitochondrial promoters HSP1 and mitochondrial extracts. LSP highlights several similarities. Transcription at these sites Next, we analyzed mutations of individual bases between the initiates at adenosine residues after a short cytosine-rich stretch polycytosine tract and the coding sequence of 12S. The T642G (Fig. 2A). A similar motif is found at the site of HSP2 initiation. mutation potently induced transcription by recombinant tran- Determination of the 5′ end of HSP2 transcripts using RNA scription factors (Fig. 3C). Other substitutions near the site of obtained from tissues or intact mitochondria is complicated by the initiation caused modest but reproducible alterations in RNA proximity of the promoter to the 5′ end of the mature 12S rRNA, synthesis. Because substitutions may cause initiation at ectopic which is present in vast excess. The examination of transcripts sites, we used S1 nuclease analysis to examine the size of the produced using recombinant POLRMT, TFB2M, and TFAM transcript produced from the T642G template and found that it allows
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