Structural Basis of Mitochondrial Transcription Initiation.

Transcription in human mitochondria is driven by a single-subunit, factor-dependent RNA polymerase (mtRNAP). Despite its critical role in both expression and replication of the mitochondrial genome, transcription initiation by mtRNAP remains poorly understood. Here, we report crystal structures of human mitochondrial transcription initiation complexes assembled on both light and heavy strand promoters. The structures reveal how transcription factors TFAM and TFB2M assist mtRNAP to achieve promoter-dependent initiation. TFAM tethers the N-terminal region of mtRNAP to recruit the polymerase to the promoter whereas TFB2M induces structural changes in mtRNAP to enable promoter opening and trapping of the DNA non-template strand. Structural comparisons demonstrate that the initiation mechanism in mitochondria is distinct from that in the well-studied nuclear, bacterial, or bacteriophage transcription systems but that similarities are found on the topological and conceptual level. These results provide a framework for studying the regulation of gene expression and DNA replication in mitochondria.

Mechanism of Transcription Anti-termination in Human Mitochondria.

In human mitochondria, transcription termination events at a G-quadruplex region near the replication origin are thought to drive replication of mtDNA by generation of an RNA primer. This process is suppressed by a key regulator of mtDNA-the transcription factor TEFM. We determined the structure of an anti-termination complex in which TEFM is bound to transcribing mtRNAP. The structure reveals interactions of the dimeric pseudonuclease core of TEFM with mobile structural elements in mtRNAP and the nucleic acid components of the elongation complex (EC). Binding of TEFM to the DNA forms a downstream "sliding clamp," providing high processivity to the EC. TEFM also binds near the RNA exit channel to prevent formation of the RNA G-quadruplex structure required for termination and thus synthesis of the replication primer. Our data provide insights into target specificity of TEFM and mechanisms by which it regulates the switch between transcription and replication of mtDNA.

Cryo-EM Structures Reveal Transcription Initiation Steps by Yeast Mitochondrial RNA Polymerase.

Mitochondrial RNA polymerase (mtRNAP) is crucial in cellular energy production, yet understanding of mitochondrial DNA transcription initiation lags that of bacterial and nuclear DNA transcription. We report structures of two transcription initiation intermediate states of yeast mtRNAP that explain promoter melting, template alignment, DNA scrunching, abortive synthesis, and transition into elongation. In the partially melted initiation complex (PmIC), transcription factor MTF1 makes base-specific interactions with flipped non-template (NT) nucleotides "AAGT" at -4 to -1 positions of the DNA promoter. In the initiation complex (IC), the template in the expanded 7-mer bubble positions the RNA and NTP analog UTPαS, while NT scrunches into an NT loop. The scrunched NT loop is stabilized by the centrally positioned MTF1 C-tail. The IC and PmIC states coexist in solution, revealing a dynamic equilibrium between two functional states. Frequent scrunching/unscruching transitions and the imminent steric clashes of the inflating NT loop and growing RNA:DNA with the C-tail explain abortive synthesis and transition into elongation.

The C-terminal tails of the mitochondrial transcription factors Mtf1 and TFB2M are part of an autoinhibitory mechanism that regulates DNA binding.

The structurally homologous Mtf1 and TFB2M proteins serve as transcription initiation factors of mitochondrial RNA polymerases in Saccharomyces cerevisiae and humans, respectively. These transcription factors directly interact with the nontemplate strand of the transcription bubble to drive promoter melting. Given the key roles of Mtf1 and TFB2M in promoter-specific transcription initiation, it can be expected that the DNA binding activity of the mitochondrial transcription factors is regulated to prevent DNA binding at inappropriate times. However, little information is available on how mitochondrial DNA transcription is regulated. While studying C-terminal (C-tail) deletion mutants of Mtf1 and TFB2M, we stumbled upon a finding that suggested that the flexible C-tail region of these factors autoregulates their DNA binding activity. Quantitative DNA binding studies with fluorescence anisotropy-based titrations revealed that Mtf1 with an intact C-tail has no affinity for DNA but deletion of the C-tail greatly increases Mtf1's DNA binding affinity. Similar observations were made with TFB2M, although autoinhibition by the C-tail of TFB2M was not as complete as in Mtf1. Analysis of available TFB2M structures disclosed that the C-tail engages in intramolecular interactions with the DNA binding groove in the free factor, which, we propose, inhibits its DNA binding activity. Further experiments showed that RNA polymerase relieves this autoinhibition by interacting with the C-tail and engaging it in complex formation. In conclusion, our biochemical and structural analyses reveal autoinhibitory and activation mechanisms of mitochondrial transcription factors that regulate their DNA binding activities and aid in specific assembly of transcription initiation complexes.

Phosphorylation of mitochondrial transcription factor B2 controls mitochondrial DNA binding and transcription.

Mammalian cells contain genetic information in two compartments, the nucleus and the mitochondria. Mitochondrial gene expression must be coordinated with nuclear gene expression to respond to cellular energetic needs. To gain insight into the coordination between the nucleus and mitochondria, there is a need to understand the regulation of transcription of mitochondrial DNA (mtDNA). Reversible protein post-translational modifications of the mtDNA transcriptional machinery may be one way to control mtDNA transcription. Here we focus on a member of the mtDNA transcription initiation complex, mitochondrial transcription factor B2 (TFB2M). TFB2M melts mtDNA at the promoter to allow the RNA polymerase (POLRMT) to access the DNA template and initiate transcription. Three phosphorylation sites have been previously identified on TFB2M by mass spectrometry: threonine 184, serine 197, and threonine 313. Phosphomimetics were established at these positions. Proteins were purified and analyzed for their ability to bind mtDNA and initiate transcription in vitro. Our results indicate phosphorylation at threonine 184 and threonine 313 impairs promoter binding and prevents transcription. These findings provide a potential regulatory mechanism of mtDNA transcription and help clarify the importance of protein post-translational modifications in mitochondrial function.

Over-expression of TFB2M facilitates cell growth and metastasis via activating ROS-Akt-NF-κB signalling in hepatocellular carcinoma.

BACKGROUND & AIMS: Human TFB2M (mitochondrial transcription factor B2) is a key regulator of mitochondria transcription. Our bioinformatic analysis based on the cancer genome atlas (TCGA) data revealed an aberrant over-expression of TFB2M in hepatocellular carcinoma (HCC). However, the functional roles of TFB2M in tumourigenesis remains unexplored, including HCC. METHODS: The expression and clinical significance of TFB2M were evaluated by qRT-PCR and western blot analysis. The biological effects and underlying mechanisms of TFB2M in HCC were determined by cell proliferation, colony formation, cell cycle, apoptosis, migration and invasion assays. RESULTS: TFB2M was commonly up-regulated in HCC mainly because of the down-regulation of miR101-3p, which significantly correlated with poor survival of HCC patients. Functional experiments revealed that TFB2M significantly promoted HCC cell proliferation, migration and invasion, while inhibited apoptosis in vitro and promoted xenograft tumourigenesis and lung metastasis in nude mice models in vivo. Mechanistically, increased production of reactive oxygen species (ROS) and subsequently activated Akt/NF-κB signalling was found to be involved in the promotion of growth and metastasis by TFB2M in HCC cells. CONCLUSIONS: These findings suggest that TFB2M plays a pivotal oncogenic role in HCC cells through activating ROS-Akt-NF-κB signalling pathway.

Identification of a rare homozygous c.790C>T variation in the TFB2M gene in Korean patients with autism spectrum disorder.

Mitochondrial dysfunction and subsequent enhanced oxidative stress is implicated in the pathogenesis of autism spectrum disorder (ASD). Mitochondrial transcription factor B2 (TFB2M) is an essential protein in mitochondrial gene expression. No reports have described TFB2M mutations and variations involved in any human diseases. We identified a rare homozygous c.790C>T (His264Tyr) variation in TFB2M gene in two Korean siblings with ASD by whole-exome sequencing. The roles of the TFB2M variation in the pathogenesis of ASD were investigated. Patient fibroblasts revealed increased transcription of mitochondrial genes and mitochondrial function in terms of ATP, membrane potential, oxygen consumption, and reactive oxygen species (ROS). Overexpression of the TFB2M variant in primary-cultured fibroblasts demonstrated significantly increased transcription of mitochondrial genes and mitochondrial function compared with overexpression of wild-type TFB2M. Molecular dynamics simulation of the TFB2M variant protein suggested an increase in the rigidity of the hinge region, which may cause alterations in loading and/or unloading of TFB2M on target DNA. Our results suggest that augmentation of mitochondrial gene expression and subsequent enhancement of mitochondrial function may be associated with the pathogenesis of ASD in Korean patients.

Human Mitochondrial Transcription Initiation Complexes Have Similar Topology on the Light and Heavy Strand Promoters.

Transcription is a highly regulated process in all domains of life. In human mitochondria, transcription of the circular genome involves only two promoters, called light strand promoter (LSP) and heavy strand promoter (HSP), located in the opposite DNA strands. Initiation of transcription occurs upon sequential assembly of an initiation complex that includes mitochondrial RNA polymerase (mtRNAP) and the initiation factors mitochondrial transcription factor A (TFAM) and TFB2M. It has been recently suggested that the transcription initiation factor TFAM binds to HSP and LSP in opposite directions, implying that the mechanisms of transcription initiation are drastically dissimilar at these promoters. In contrast, we found that binding of TFAM to HSP and the subsequent recruitment of mtRNAP results in a pre-initiation complex that is remarkably similar in topology and properties to that formed at the LSP promoter. Our data suggest that assembly of the pre-initiation complexes on LSP and HSP brings these transcription units in close proximity, providing an opportunity for regulatory proteins to simultaneously control transcription initiation in both mtDNA strands.

Structural models of mammalian mitochondrial transcription factor B2.

Mammalian mitochondrial DNA (mtDNA) encodes 13 core proteins of oxidative phosphorylation, 12S and 16S ribosomal RNAs, and 22 transfer RNAs. Mutations and deletions of mtDNA and/or nuclear genes encoding mitochondrial proteins have been implicated in a wide range of diseases. Thus, cell survival and health of the organism require some steady-state level of the mitochondrial genome and its expression. In mammalian systems, the mitochondrial transcription factor B2 (mtTFB2 or TFB2M) is indispensable for transcription initiation. TFB2M along with two other proteins, mitochondrial RNA polymerase (mtRNAP or POLRMT) and mitochondrial transcription factor A (mtTFA or TFAM), are key components of the core mitochondrial transcription apparatus. Structural information for POLRMT and TFAM from humans is available; however, there is no available structure for TFB2M. In the present study, three-dimensional structure of TFB2M from humans was modeled using a combination of homology modeling and small-angle X-ray scattering (SAXS). The TFB2M structural model adds substantively to our understanding of TFB2M function. An explanation for the low or absent RNA methyltransferase activity is provided. A putative nucleic acid-binding site is revealed. The amino and carboxy termini, while likely lacking defined secondary structure, appear to adopt compact, globular conformations, thus "capping" the ends of the protein. Finally, sites of interaction of TFB2M with other factors, protein and/or nucleic acid, are suggested by the identification of species-specific clusters on the surface of the protein.

Expression and Purification of Recombinant Human Mitochondrial RNA Polymerase (POLRMT) and the Initiation Factors TFAM and TFB2M.

Human mitochondrial DNA (mtDNA) encodes several components of oxidative phosphorylation responsible for the bulk of cellular energy production. The mtDNA is transcribed by a dedicated human mitochondrial RNA polymerase (POLRMT) that is structurally distinct from its nuclear counterparts, instead closely resembling the single-subunit viral RNA polymerases (e.g., T7 RNA polymerase). The initiation of transcription by POLRMT is aided by two initiation factors: transcription factor A, mitochondrial (TFAM), and transcription factor B2, mitochondrial (TFB2M). Although many details of human mitochondrial transcription initiation have been elucidated with in vitro biochemical and structural studies, much remains to be addressed relating to the mechanism and regulation of transcription. Studies of such mechanisms require reliable, high-yield, and high-purity methods for protein production, and this protocol provides the level of detail and troubleshooting tips that are necessary for a novice to generate meaningful amounts of proteins for experimental work. The current protocol describes how to purify recombinant POLRMT, TFAM, and TFB2M from Escherichia coli using techniques such as affinity column chromatography (Ni2+ and heparin), how to remove the solubility tags with TEV protease and recover untagged proteins of interest, and how to overcome commonly encountered challenges in obtaining high yield of each protein. Key features • This protocol builds upon purification methods developed by Patel lab (Ramachandran et al., 2017) and others with greater detail than previously published works. • The protocol requires several days to complete as various steps are designed to be performed overnight. • The recombinantly purified proteins have been successfully used for in vitro transcription experiments, allowing for finer control of experimental components in a minimalistic system.

In Vitro Reconstitution of Human Mitochondrial Transcription.

In vitro assay based on a reconstituted mitochondrial transcription system serves as a method of choice to probe the functional importance of proteins and their structural motifs. Here we describe protocols for transcription assays designed to probe activity of the human mitochondrial RNA polymerase and the transcription initiation complex using RNA-DNA scaffold and synthetic promoter templates.

Mechanisms of mammalian mitochondrial transcription.

Numerous age-related human diseases have been associated with deficiencies in cellular energy production. Moreover, genetic alterations resulting in mitochondrial dysfunction are the cause of inheritable disorders commonly known as mitochondrial diseases. Many of these deficiencies have been directly or indirectly linked to deficits in mitochondrial gene expression. Transcription is an essential step in gene expression and elucidating the molecular mechanisms involved in this process is critical for understanding defects in energy production. For the past five decades, substantial efforts have been invested in the field of mitochondrial transcription. These efforts have led to the discovery of the main protein factors responsible for transcription as well as to a basic mechanistic understanding of the transcription process. They have also revealed various mechanisms of transcriptional regulation as well as the links that exist between the transcription process and downstream processes of RNA maturation. Here, we review the knowledge gathered in early mitochondrial transcription studies and focus on recent findings that shape our current understanding of mitochondrial transcription, posttranscriptional processing, as well as transcriptional regulation in mammalian systems.

Topological requirements of the mitochondrial heavy-strand promoters.

In vitro studies of mitochondrial transcription often use linear templates that fail to replicate key features of transcription on a circular genome. We developed a plasmid-based system for the analysis of heavy-strand promoters that recapitulates key features of native mtDNA to study topological and protein requirements of promoter activation. The heavy-strand promoters (HSP1 and HSP2) are simultaneously active on a circular template. HSP2 requires supercoiling for maximal activation. Increasing TFAM concentrations suppress HSP2 at levels that result in HSP1 stimulation. This study shows distinct modes of promoter activation, providing opportunities for the regulation of mitochondrial gene expression by promoter selection.

Transcribing ß-cell mitochondria in health and disease.

BACKGROUND: The recent genome-wide association studies (GWAS) of Type 2 Diabetes (T2D) have identified the pancreatic ß-cell as the culprit in the pathogenesis of the disease. Mitochondrial metabolism plays a crucial role in the processes controlling release of insulin and ß-cell mass. This notion implies that mechanisms controlling mitochondrial function have the potential to play a decisive pathogenetic role in T2D. SCOPE OF THE REVIEW: This article reviews studies demonstrating that there is indeed mitochondrial dysfunction in islets in T2D, and that GWAS have identified a variant in the gene encoding transcription factor B1 mitochondrial (TFB1M), predisposing to T2D due to mitochondrial dysfunction and impaired insulin secretion. Mechanistic studies of the nature of this pathogenetic link, as well as of other mitochondrial transcription factors, are described. MAJOR CONCLUSIONS: Based on this, it is argued that transcription and translation in mitochondria are critical processes determining mitochondrial function in ß-cells in health and disease.