Yaroslav I. Morozov

Structure of human mitochondrial RNA polymerase

Transcription of the mitochondrial genome is performed by a single-subunit RNA polymerase (mtRNAP) that is distantly related to the RNAP of bacteriophage T7, the pol I family of DNA polymerases, and single-subunit RNAPs from chloroplasts. Whereas T7 RNAP can initiate transcription by itself, mtRNAP requires the factors TFAM and TFB2M for binding and melting promoter DNA. TFAM is an abundant protein that binds and bends promoter DNA 15–40 base pairs upstream of the transcription start site, and stimulates the recruitment of mtRNAP and TFB2M to the promoter. TFB2M assists mtRNAP in promoter melting and reaches the active site of mtRNAP to interact with the first base pair of the RNA–DNA hybrid. Here we report the X-ray structure of human mtRNAP at 2.5 Å resolution, which reveals a T7-like catalytic carboxy-terminal domain, an amino-terminal domain that remotely resembles the T7 promoter-binding domain, a novel pentatricopeptide repeat domain, and a flexible N-terminal extension. The pentatricopeptide repeat domain sequesters an AT-rich recognition loop, which binds promoter DNA in T7 RNAP, probably explaining the need for TFAM during promoter binding. Consistent with this, substitution of a conserved arginine residue in the AT-rich recognition loop, or release of this loop by deletion of the N-terminal part of mtRNAP, had no effect on transcription. The fingers domain and the intercalating hairpin, which melts DNA in phage RNAPs, are repositioned, explaining the need for TFB2M during promoter melting. Our results provide a new venue for the mechanistic analysis of mitochondrial transcription. They also indicate how an early phage-like mtRNAP lost functions in promoter binding and melting, which were provided by initiation factors in trans during evolution, to enable mitochondrial gene regulation and the adaptation of mitochondrial function to changes in the environment.

Phosphorylation of human TFAM in mitochondria impairs DNA binding and promotes degradation by the AAA+ Lon protease.

Human mitochondrial transcription factor A (TFAM) is a high-mobility group (HMG) protein at the nexus of mitochondrial DNA (mtDNA) replication, transcription, and inheritance. Little is known about the mechanisms underlying its posttranslational regulation. Here, we demonstrate that TFAM is phosphorylated within its HMG box 1 (HMG1) by cAMP-dependent protein kinase in mitochondria. HMG1 phosphorylation impairs the ability of TFAM to bind DNA and to activate transcription. We show that only DNA-free TFAM is degraded by the Lon protease, which is inhibited by the anticancer drug bortezomib. In cells with normal mtDNA levels, HMG1-phosphorylated TFAM is degraded by Lon. However, in cells with severe mtDNA deficits, nonphosphorylated TFAM is also degraded, as it is DNA free. Depleting Lon in these cells increases levels of TFAM and upregulates mtDNA content, albeit transiently. Phosphorylation and proteolysis thus provide mechanisms for rapid fine-tuning of TFAM function and abundance in mitochondria, which are crucial for maintaining and expressing mtDNA.

Replication-transcription switch in human mitochondria

Switching transcription and replication Because mitochondrial DNA is circular, the transcription and replication machinery might be expected to collide. A single mitochondrial RNA polymerase (mtRNAP) transcribes the mitochondrial DNA and also generates primers for replication. Agaronyan et al. now show that transcription and replication are kept separate in human mitochondria, with the mitochondrial transcription elongation factor TEFM serving as a key player in the switch. In the absence of TEFM, mtRNAP terminates downstream from the promoter, forming primers to promote replication. In the presence of TEFM, the primers are not formed, and the overall processivity of mtRNAP elongation complexes is enhanced, promoting genome transcription. These mutually exclusive mechanisms allow the processes to proceed independently as needed by the cell. Science, this issue p. 548 Human mitochondrial elongation factor determines whether mitochondrial DNA will be copied or transcribed. Coordinated replication and expression of the mitochondrial genome is critical for metabolically active cells during various stages of development. However, it is not known whether replication and transcription can occur simultaneously without interfering with each other and whether mitochondrial DNA copy number can be regulated by the transcription machinery. We found that interaction of human transcription elongation factor TEFM with mitochondrial RNA polymerase and nascent transcript prevents the generation of replication primers and increases transcription processivity and thereby serves as a molecular switch between replication and transcription, which appear to be mutually exclusive processes in mitochondria. TEFM may allow mitochondria to increase transcription rates and, as a consequence, respiration and adenosine triphosphate production without the need to replicate mitochondrial DNA, as has been observed during spermatogenesis and the early stages of embryogenesis.

Structure of human mitochondrial RNA polymerase elongation complex

Here we report the crystal structure of the human mitochondrial RNA polymerase (mtRNAP) transcription elongation complex, determined at 2.65-Å resolution. The structure reveals a 9-bp hybrid formed between the DNA template and the RNA transcript and one turn of DNA both upstream and downstream of the hybrid. Comparisons with the distantly related RNA polymerase (RNAP) from bacteriophage T7 indicates conserved mechanisms for substrate binding and nucleotide incorporation but also strong mechanistic differences. Whereas T7 RNAP refolds during the transition from initiation to elongation, mtRNAP adopts an intermediary conformation that is capable of elongation without refolding. The intercalating hairpin that melts DNA during T7 RNAP initiation separates RNA from DNA during mtRNAP elongation. Newly synthesized RNA exits toward the pentatricopeptide repeat (PPR) domain, a unique feature of mtRNAP with conserved RNA-recognition motifs.

A novel intermediate in transcription initiation by human mitochondrial RNA polymerase

The mitochondrial genome is transcribed by a single-subunit T7 phage-like RNA polymerase (mtRNAP), structurally unrelated to cellular RNAPs. In higher eukaryotes, mtRNAP requires two transcription factors for efficient initiation—TFAM, a major nucleoid protein, and TFB2M, a transient component of mtRNAP catalytic site. The mechanisms behind assembly of the mitochondrial transcription machinery and its regulation are poorly understood. We isolated and identified a previously unknown human mitochondrial transcription intermediate—a pre-initiation complex that includes mtRNAP, TFAM and promoter DNA. Using protein–protein cross-linking, we demonstrate that human TFAM binds to the N-terminal domain of mtRNAP, which results in bending of the promoter DNA around mtRNAP. The subsequent recruitment of TFB2M induces promoter melting and formation of an open initiation complex. Our data indicate that the pre-initiation complex is likely to be an important target for transcription regulation and provide basis for further structural, biochemical and biophysical studies of mitochondrial transcription.

A model for transcription initiation in human mitochondria

Regulation of transcription of mtDNA is thought to be crucial for maintenance of redox potential and vitality of the cell but is poorly understood at the molecular level. In this study we mapped the binding sites of the core transcription initiation factors TFAM and TFB2M on human mitochondrial RNA polymerase, and interactions of the latter with promoter DNA. This allowed us to construct a detailed structural model, which displays a remarkable level of interaction between the components of the initiation complex (IC). The architecture of the mitochondrial IC suggests mechanisms of promoter binding and recognition that are distinct from the mechanisms found in RNAPs operating in all domains of life, and illuminates strategies of transcription regulation developed at the very early stages of evolution of gene expression.

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.

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.

Single-column purification of the tag-free, recombinant form of the neuronal calcium sensor protein, hippocalcin expressed in Escherichia coli.

Hippocalcin is a 193 aa protein that is a member of the neuronal calcium sensor protein family, whose functions are regulated by calcium. Mice that lack the function of this protein are compromised in the long term potentiation aspect of memory generation. Recently, mutations in the gene have been linked with dystonia in human. The protein has no intrinsic enzyme activity but is known to bind to variety of target proteins. Very little information is available on how the protein executes its critical role in signaling pathways, except that it is regulated by binding of calcium. Further delineation of its function requires large amounts of pure protein. In this report, we present a single-step purification procedure that yields high quantities of the bacterially expressed, recombinant protein. The procedure may be adapted to purify the protein from inclusion bodies or cytosol in its myristoylated or non-myristoylated forms. MALDI-MS (in source decay) analyses demonstrates that the myristoylation occurs at the glycine residue. The protein is also biologically active as measured through tryptophan fluorescence, mobility shift and guanylate cyclase activity assays. Thus, further analyses of hippocalcin, both structural and functional, need no longer be limited by protein availability.

Data on the identity and myristoylation state of recombinant, purified hippocalcin.

In this data article we report on the purity and post translation modification of bacterially expressed and purified recombinant hippocalcin (HPCA): a member of the neuronal calcium sensor protein family, whose functions are regulated by calcium. MALDI-TOF in source decay (ISD) analysis was used to identify both the myristoylated or non-myristoylated forms of the protein. MALDI-TOF ISD data on the identity of the protein, amino acid sequence and myristoylation efficiency are provided. This data relates to the article “Single-Column Purification of the Tag-free, Recombinant Form of the Neuronal Calcium Sensor Protein, Hippocalcin Expressed in Eschericia coli” [1].