Core human mitochondrial transcription apparatus is a regulated two-component system in vitro

Abstract

The core human mitochondrial transcription apparatus is currently regarded as an obligate three-component system comprising the bacteriophage T7-related mitochondrial RNA polymerase, the rRNA methyltransferase-related transcription factor, h-mtTFB2, and the high mobility group box transcription/DNA-packaging factor, h-mtTFA/TFAM. Using a faithful recombinant human mitochondrial transcription system from Escherichia coli, we demonstrate that specific initiation from the mtDNA promoters, LSP and HSP1, only requires mitochondrial RNA polymerase and h-mtTFB2 in vitro. When h-mtTFA is added to these basal components, LSP exhibits a much lower threshold for activation and a larger amplitude response than HSP1. In addition, when LSP and HSP1 are together on the same transcription template, h-mtTFA-independent transcription from HSP1 and h-mtTFA-dependent transcription from both promoters is enhanced and a higher concentration of h-mtTFA is required to stimulate HSP1. Promoter competition experiments revealed that, in addition to LSP competing transcription components away from HSP1, additional cis-acting signals are involved in these aspects of promoter regulation. Based on these results, we speculate that the human mitochondrial transcription system may have evolved to differentially regulate transcription initiation and transcription-primed mtDNA replication in response to the amount of h-mtTFA associated with nucleoids, which could begin to explain the heterogeneity of nucleoid structure and activity in vivo. Furthermore, this study sheds new light on the evolution of mitochondrial transcription components by showing that the human system is a regulated two-component system in vitro, and thus more akin to that of budding yeast than thought previously.

Fig. 1.

A recombinant human mitochondrial transcription system from E.coli recapitulates promoter-specific initiation from linear DNA templates containing both LSP and HSP1. (A) Schematic representation of the D-loop regulatory region of human mtDNA, with salient regulatory elements indicated, including the hallmark D-loop strand (thick black bent arrow). The LSP, HSP1, and HSP2 are depicted as thin bent arrows. Known binding sites for h-mtTFA at LSP and HSP1 are indicated as black and gray boxes, respectively, and conserved sequence blocks (CSB) 1, 2, and 3 are depicted as white boxes. The solid lines below the D-loop region diagram denote the linear mtDNA templates, LSP3 and LSP3.1, used in the run-off transcription reactions in B and C, respectively. The major RNA transcripts observed in the in vitro reactions are indicated by the numbered (1–3) or lettered (A and B) dashed lines. From the template LSP3, transcript 1 is the full-length transcript initiated at HSP1, transcript 2 is the full-length transcript initiated at LSP, and transcript 3 is the truncated product resulting from transcription termination/stalling downstream of CSB2. From template LSP3.1, transcript A is the full-length transcript initiated at HSP1 and transcript B is full-length transcript initiated at LSP. Predicted transcript sizes are 267, 182, and 118 nucleotides for transcripts 1, 2, and 3, respectively, and 280 and 97 nucleotides for transcripts A and B, respectively. (B) Autoradiogram of transcription products obtained from a typical in vitro-transcription reaction is shown: POLRMT (16 nM), h-mtTFB2 (16 nM), h-mtTFA (10 nM), and template LSP3 (3.4 nM) are run next to a labeled 10 nt DNA ladder, with the sizes indicated. The slight disparity between observed and predicted sizes of the transcripts is due to different mobility of RNA versus DNA in these gels. (C) Same as (B), except the template LSP3.1 was used to eliminate production of the truncated product at CSB2 and h-mtTFB2 was present at 80 nM.

Fig. 2.

Promoter-specific initiation of transcription at HSP1 and LSP by POLRMT and h-mtTFB2 in the absence of h-mtTFA. (A) Autoradiogram of transcription products obtained in run-off transcription reactions using template LSP3.1 (see Fig. 1A) and POLRMT (16 nM) either alone (lane 1) or together with h-mtTFA (4 nM; lane 2), h-mtTFB2 (16 nM; lane 3), or both (lane 4). (B) Autoradiogram of reactions from the LSP3 template and using POLRMT (16 nM) and increasing concentration of h-mtTFB2 (16, 32, 48, and 64 nM), lanes 1–4. Lane 5 is the same as lane 1, but also includes 10 nM h-mtTFA added to the reaction. (C) Autoradiogram of products obtained from h-mtTFA-dependent transcription reactions (POLRMT, 16 nM; h-mtTFA, 10 nM; h-mtTFB2, 64 nM) that were incubated in the presence (+) or absence (-) anti-h-mtTFA antibody (α-mtTFA) as indicated, using the LSP3 template.

Fig. 3.

Differential dose response of HSP1 and LSP transcription to increasing amounts of h-mtTFA. Shown is an autoradiogram of representative transcripts produced in response to increasing concentrations of h-mtTFA (4, 20, 40, 200, 400, 600, and 800 nM per reaction) added to the standard transcription reaction containing POLRMT (16 nM), h-mtTFB2 (16 nM), and LSP3.1 (3.4 nM). The major full-length LSP and HSP1 transcripts are labeled on the left. Reactions were performed in triplicate (Fig. S2), products were quantified by phosphorimager analysis (correcting for UTP incorporation) and the mean +/- one standard deviation is plotted. Lane 1 contains a labeled DNA ladder (L).

Fig. 4.

Isolated LSP and HSP1 templates support specific h-mtTFA-independent transcription initiation, but promoter responses to h-mtTFA are altered when both promoters are present on the same template. (A) Shown are results from RNA-trinucleotide-primed transcription reactions performed on three different oligonucleotide templates: an isolated LSP, isolated HSP1, and a dual-promoter template (similar to LSP3.1, but with less DNA downstream of each promoter; see Methods and Materials for details). Isolated promoter reactions contained double-stranded oligonucleotide DNA template (100 nM), POLRMT (100 nM), and h-mtTFB2 (100 nM) without (0) or with increasing amounts of h-mtTFA added (25, 50, 100, 200, 500, 1,000, and 2,000 nM). Dual-promoter reactions were the same, except POLRMT and h-mtTFB2 were each present at 200 nM. The predicted product from each of the isolated LSP and HSP1 templates is 40 nt. The predicted products from the dual-promoter template are 45 nt (HSP1) and 35 nt (LSP). Lanes labeled B are blank. The left lane contains labeled 10 bp ladder with sizes indicated on the left. (B) Quantitation of dose response to h-mtTFA shown in part A where the amount (μM) of RNA product produced is plotted as a function of h-mtTFA concentration (error bars denote +/- one standard error from the mean).