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.

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 transcription 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.

Fig. 2.

Localization of the HSP2 start site. (A) Alignment of the mitochondrial promoters. The polycytosine tract preceding transcription is highlighted in bold, and the primary transcript is underlined. The HSP2 start site as drawn is taken from the work by Martin et al. (7). (B) S1 analysis of RNA produced by recombinant TFAM, TFB2, and POLRMT acting on the nucleotides 583–801 template (Left) produced bands corresponding to ends between positions 642 and 644. For convenience, the sequencing ladder shown reads the nontemplate strand and has the same 5′ end as the S1 probe. A small amount of fully protected probe is also seen because of synthesis of full-length RNA from linear templates. We examined RNA produced from the nucleotides 460–801 template (Right) using an S1 probe complementary to nucleotides 554–607. S1 cleavage of this probe gave a 47-nt product matching the expected size of the HSP1 transcript. (C) RACE sequencing from RNA synthesized by recombinant proteins transcribing nucleotides 583–801. Sequences inserted by the reverse transcriptase (polycytosine) predominate upstream of A644, indicating that 5′ ends begin predominantly at this point.

Fig. 3.

Mutagenesis of HSP2. (A) A series of mutation constructs were created in which blocks of upstream nucleotides where replaced by poly-T tracts. (B) Transcription by HeLa cell mitochondrial extracts (10 μg) in the presence of a 100-nM nucleotides 460–801 template with the specified poly-T replacement mutations. (C) A series of HSP2 promoter mutants were created in which one base at a time was mutated from the WT sequence. The ability of HSP2 mutants to support transcription was investigated using recombinant proteins as described previously. The WT sequence is noted above the autoradiogram.

Fig. 4.

HSP2 transcription responds to TFAM dosage. Transcription reactions were performed using the indicated concentration of TFAM. Template, POLRMT, and TFB2M were added at 100 nM.

Fig. 5.

Heavy-strand promoters have differential responses to ATP concentration. Transcription reactions were performed using the nucleotides 460–801 template containing both the HSP1 and HSP2 promoters and HeLa cell mitochondrial extracts. All components were held constant except for the level of ATP, which varied up to 5 mM.