Structure, mechanism, and regulation of mitochondrial DNA transcription initiation

Abstract

Mitochondria are specialized compartments that produce requisite ATP to fuel cellular functions and serve as centers of metabolite processing, cellular signaling, and apoptosis. To accomplish these roles, mitochondria rely on the genetic information in their small genome (mitochondrial DNA) and the nucleus. A growing appreciation for mitochondria's role in a myriad of human diseases, including inherited genetic disorders, degenerative diseases, inflammation, and cancer, has fueled the study of biochemical mechanisms that control mitochondrial function. The mitochondrial transcriptional machinery is different from nuclear machinery. The in vitro re-constituted transcriptional complexes of Saccharomyces cerevisiae (yeast) and humans, aided with high-resolution structures and biochemical characterizations, have provided a deeper understanding of the mechanism and regulation of mitochondrial DNA transcription. In this review, we will discuss recent advances in the structure and mechanism of mitochondrial transcription initiation. We will follow up with recent discoveries and formative findings regarding the regulatory events that control mitochondrial DNA transcription, focusing on those involved in cross-talk between the mitochondria and nucleus.

Keywords: DNA transcription; RNA polymerase; enzyme mechanism; enzyme structure; human mitochondrial RNA polymerase; mitochondria; mitochondrial DNA (mtDNA); mitochondrial DNA transcription; mitochondrial gene regulation; structure-function; transcription; transcription initiation factors; transcription regulation; yeast mitochondrial RNA polymerase.

Figure 1

Human mitochondrial DNA composition. Human mtDNA is depicted with a heavy strand in black and light strand in gray. rRNAs (yellow), mRNAs (blue), and tRNAs (green) are labeled. Transcription is bidirectional and initiated in the D-loop control region (shown expanded) from three promoters, HSP1, HSP2, and LSP. TFAM (pale green) binds mtDNA upstream of promoters, recruiting TFB2M (orange) and h-mtRNAP (gray) to initiate transcription.

Figure 2

Comparison of single-subunit RNAP promoters and protein structures.A, the DNA promoter sequence (nontemplate strand) of phage T7, yeast, and human mtDNA. The conserved nucleotides within the promoter region are in boldface type. T7 promoters are conserved from −17 to +2, y-mtDNA promoters are conserved from −8 to +1, and h-mtDNA promoters have conserved −7, −3, +1 to +3, and +5 base pairs. B, the domain structures of T7 RNAP, y-mtRNAP, and h-mtRNAP. The color-coded regions show conserved elements in the CTD and the NTD. An N-terminal extension (NTE) is present in mtRNAPs but lacking in T7 RNAP. C, high-resolution structures of the initiation complexes of T7 RNAP with 3-bp RNA:DNA (PDB entry 1QLN), yeast mtRNAP with 2-bp RNA:DNA and the next NTP (PDB entry 6YMW), and human mtRNAP without NTP (PDB entry 6ERP) are shown. The conserved elements in the three RNAPs are color-coded and labeled. The template DNA is shown in blue, nontemplate DNA in cyan, and RNA in magenta. The Y-helix and O-helix in the fingers domain in y-mtRNAP are labeled as Y and O, respectively.

Figure 4

Detailed views of the active-site cavity in the initiation complexes of yeast mtRNAP.A, view into the active-site cavity of the IC₂+NTP complex showing the scrunched nontemplate strand (cyan), the melted template strand (blue) aligned with the 2-mer RNA (magenta), the next incoming NTP (red), and the catalytic metal ion (green). The conserved elements, including the thumb domain (green), ICH (gray), MTF1 C-tail (orange), and palm domain (salmon pink), are stabilizing the melted template and nontemplate DNA strands in the active site. B, the MTF1 C-tail is highlighted to show its proximity to the 5′-end of the RNA:DNA hybrid and the scrunched NT-loop. The C-tail is expected to sterically clash (black arrows) with the RNA:DNA and NT-loop. C, the partially melted initiation complex (PmIC) shows the flipped −4 to −1 bases of the melted nontemplate strand interacting with the ICH of y-mtRNAP and MTF1. D, a detailed view of the base-stacking and base-specific interactions of the −2 G of the nontemplate strand with the residues of MTF1. E, an in-depth look of the active site of IC₂+NTP with 2-mer RNA and incoming NTP interactions with the fingers (O-helix) and palm domain residues. The Mg²⁺ (green) in the structure is coordinated with the NTP and residues of the palm domain.

Figure 3

Transcription initiation pathway of mtRNAP. Transcription initiation factor MTF1/TFB2M (in orange) equilibrates between two states: an autoinhibited state, where the flexible C-tail occludes the DNA-binding site, and a free state, where the C-tail is free to interact with the mtRNAP. The exact pathway of closed complex formation is not known. Here, MTF1/TFB2M is shown to associate with a DNA-bound mtRNAP (in gray) to form a closed complex, in which DNA is slightly bent but not melted (template in blue, nontemplate in green). In the h-mtRNAP complex, TFAM (not shown here) would be bound to the upstream DNA assisting in promoter-specific binding. Studies of y-mtRNAP indicate an intermediate, PmIC, between the closed complex and initiation complex. In the PmIC state, base pairs from position −4 to −1 melt, MTF1/TFB2M stabilizes the bubble by interacting with the nontemplate strand, and mtRNAP interacts with the template strand. Subsequently, +1 and +2 base pairs melt to generate an IC₀ state. The MTF1/TFB2M C-tail helps position the template strand in the active site to promote binding +1 and +2 initiating NTPs in the IC₂ state. The binding of the initiating NTPs drives the conversion of PmIC to IC₂. Phosphodiester bond formation results in a 2-bp RNA:DNA hybrid, which elongates in a stepwise manner through melting of the downstream DNA and scrunching of the nontemplate strand into an NT-loop, as shown in the IC₃. The growing RNA:DNA hybrid and the NT-loop push the C-tail away from the active site cavity and help the transition into elongation after 8-nt RNA synthesis. During the transition into elongation, the promoter DNA unscrunches and unbends, and the −4 to −1 base pairs of the bubble reanneal. MTF1/TFB2M may entirely or partially dissociate during the transition into elongation. Branched pathways occur with some frequency during transcription initiation, resulting in abortive synthesis or backtracking of the mtRNAP. During abortive synthesis, the RNA transcripts in IC₂ to IC₇ dissociate into the solution; the mtRNAP remains bound to the promoter DNA in the PmIC/IC₀ state and rebinds NTPs, starting another round of transcription reaction. During backtracking, the RNA does not dissociate, but downstream DNA reanneals, fraying the 3′-end of the RNA:DNA hybrid.

Figure 5

Regulation of mitochondrial transcription overview.A, most mitochondrial proteins, including the core mtDNA transcription machinery and other nucleoid proteins, are encoded by nuclear genes, synthesized by cytosolic ribosomes, and imported into the mitochondria. Therefore, mitochondrial transcription and its regulation are largely dependent upon nuclear-encoded factors. B, a subset of these proteins associate with mtDNA, forming nucleoid particles. Nucleoid dynamics, including epigenetic modifications to mtDNA, nucleoid protein interactions, and post-translational modifications of mtDNA transcription factors, affect mtDNA accessibility and transcription. C, other nuclear-encoded mitochondrial proteins are responsible for processing nascent mtRNAs. Prior to translation, polycistronic RNAs must be cleaved, chemically modified, and adenylated to reach their mature form. D, nuclear transcription factors regulate the expression of nuclear-encoded mitochondrial proteins like TFAM and h-mtRNAP, indirectly regulating the expression of mtDNA-encoded genes. E, additionally, various canonically nuclear transcription factors translocate to the mitochondria or shuttle between the two compartments under various conditions, providing one means of coordinating mitochondrial and nuclear gene expression. F, mitochondrial factors also influence nuclear epigenetics, contributing to retrograde signaling and cross-talk between the two compartments. Finally, both the nucleus and the mitochondria sense and respond to metabolic conditions, such as nutrient availability and reactive oxygen species patterns. These broad cellular states affect gene expression in both compartments and influence signaling between the two.