Human mitochondrial transcription revisited: only TFAM and TFB2M are required for transcription of the mitochondrial genes in vitro

Abstract

Human mitochondrial transcription is driven by a single subunit RNA polymerase and a set of basal transcription factors. The development of a recombinant in vitro transcription system has allowed for a detailed molecular characterization of the individual components and their contribution to transcription initiation. We found that TFAM and TFB2M act synergistically and increase transcription efficiency 100-200-fold as compared with RNA polymerase alone. Both the light-strand promoter (LSP) and the HSP1 promoters displayed maximal levels of in vitro transcription when TFAM was present in an amount equimolar to the DNA template. Importantly, we did not detect any significant transcription activity in the presence of the TFB2M paralog, TFB1M, or when templates containing the putative HSP2 promoter were used. These data confirm previous observations that TFB1M does not function as a bona fide transcription factor and raise questions as to whether HSP2 serves as a functional promoter in vivo. In addition, we did not detect transcription stimulation by the ribosomal protein MRPL12. Thus, only two essential initiation factors, TFAM and TFB2M, and two promoters, LSP and HSP1, are required to drive transcription of the mitochondrial genome.

FIGURE 1.

TFB2M but not TFB1M is required to stimulate human mitochondrial transcription initiation. A, sequences of LSP and HSP1 aligned at their transcription start sites. Shaded boxes indicate transcripts obtained in reaction having limited sets of the substrate NTPs. B, transcription of HSP1 and LSP in the presence of POLRMT, TFAM, and TFB2M. The reactions were carried out as described under “Experimental Procedures” using ApA primer and limited NTP mixtures, and the products were resolved by 20% PAGE containing 6 m urea. C, transcription of HSP1 and LSP in the presence of POLRMT, TFAM, and TFB1M. The reactions were carried out as described above. Note that the image is overexposed (6-fold) as compared with the experiment shown in panel B to demonstrate TFAM effect on transcription by POLRMT. D, transcription of LSP and HSP1 depends on TFAM stimulation. The reactions were carried out as described above using POLRMT, TFB2M (all 50 nm), and TFAM concentrations as indicated (0–200 nm). E, single-round transcription using catalytic autolabeling. Reactions containing POLRMT (150 nm), TFB2M (150 nm), and TFAM (0–500 nm) were performed as described under “Experimental Procedures” and resolved using 4–12% Bis-Tris NuPAGE.

FIGURE 2.

Basal transcription system requires only TFAM, TFB2M, and two promoters, LSP and HSP1. A, MRPL12 does not stimulate mitochondrial transcription in vitro in the presence of the purified recombinant proteins. Reactions containing synthetic HSP1 template were performed as in Fig. 1B except that ATP, GTP, and CTP (all 0.3 mm) were used. B, MRPL12 does not stimulate transcription in the presence of mitochondrial extracts. For experiments with recombinant proteins (lanes 1–6), the individual transcription reaction mixtures contained POLRMT (400 fmol), TFB2M (400 fmol), TFAM (2.5 pmol), and the indicated mtDNA template (85 fmol). An S-100 mitochondrial lysate was used for transcription with the extracts (lanes 7–12). C, sequence of the HSP2 region. Shaded boxes indicate the 3′-terminal end of the tRNA^Phe gene and the 5′-end of 12 S RNA gene. D, run-off transcription assay using promoter templates containing LSP1, HSP1, and HSP2. Transcription was carried out in the presence of TFAM, TFB2M, and POLRMT for 40 min at 35 °C using 0.5 mm substrate NTPs. E, run-off transcription assay using plasmid templates containing LSP and HSP1/HSP2. Transcription reactions were performed as indicated in panel B. HSP1/HSP2 template was obtained by linearization of a human mtDNA fragment at position 741. Transcription of HSP1 transcription generated a 181-nt product, but no 96-nt product expected for initiation at HSP2 was observed. LSP transcription using an mtDNA fragment corresponding to positions 1–477 generated run-off and prematurely terminated (PT) products. F, transcription is initiated from HSP1, but not from HSP2, in the presence of an S-100 mitochondrial lysate. A transcription reaction using HSP1/HSP2 template (lanes 1 and 2) described above was performed in the presence of the mitochondrial extracts supplemented with TFAM (1 pmol) and TFB2M (0.25 pmol). The positions of size markers (SM) are given to the left.

FIGURE 3.

Schematics of the basal transcription initiation machinery in human mitochondria. Human mitochondrial genome contains two promoters located in the opposing DNA strands. The HSP1 promoter is responsible for synthesis of most mitochondrial genes and is activated by POLRMT-TFAM-TFB2M complex. During replication, when DNA region near oriL becomes single-stranded and forms stem-loop structure, transcription by POLRMT generates short RNA primers. This initiation event is TFAM- and TFB2M-independent. Transcription from the LSP promoter generates primers for replication at oriH as well as the rest of the tRNAs and mRNA. This initiation event, similar to HSP1, requires cooperative action of POLRMT, TFAM, and TFB2M for efficient transcription and replication.