Mechanisms of mitochondrial promoter recognition in humans and other mammalian species

Recognition of mammalian mitochondrial promoters requires the concerted action of mitochondrial RNA polymerase (mtRNAP) and transcription initiation factors TFAM and TFB2M. In this work, we found that transcript slippage results in heterogeneity of the human mitochondrial transcripts in vivo and in vitro. This allowed us to correctly interpret the RNAseq data, identify the bona fide transcription start sites (TSS), and assign mitochondrial promoters for \u3e 50% of mammalian species and some other vertebrates. The divergent structure of the mammalian promoters reveals previously unappreciated aspects of mtDNA evolution. The correct assignment of TSS also enabled us to establish the precise register of the DNA in the initiation complex and permitted investigation of the sequence-specific protein-DNA interactions. We determined the molecular basis of promoter recognition by mtRNAP and TFB2M, which cooperatively recognize bases near TSS in a species-specific manner. Our findings reveal a role of mitochondrial transcription machinery in mitonuclear coevolution and speciation

Mesoscale Structure-Function Relationships in Mitochondrial Transcriptional Condensates

In live cells, phase separation is thought to organize macromolecules into membraneless structures known as biomolecular condensates. Here, we reconstituted transcription in condensates from purified mitochondrial components using optimized in vitro reaction conditions to probe the structure-function relationships of biomolecular condensates. We find that the core components of the mt-transcription machinery form multiphasic, viscoelastic condensates in vitro. Strikingly, the rates of condensate-mediated transcription are substantially lower than in solution. The condensate-mediated decrease in transcriptional rates is associated with the formation of vesicle-like structures that are driven by the production and accumulation of RNA during transcription. The generation of RNA alters the global phase behavior and organization of transcription components within condensates. Coarse-grained simulations of mesoscale structures at equilibrium show that the components stably assemble into multiphasic condensates and that the vesicles formed in vitro are the result of dynamical arrest. Overall, our findings illustrate the complex phase behavior of transcribing, multicomponent condensates, and they highlight the intimate, bidirectional interplay of structure and function in transcriptional condensates

Structural Basis for DNA Proofreading

DNA polymerase (DNAP) can correct errors in DNA during replication by proofreading, a process critical for cell viability. However, the mechanism by which an erroneously incorporated base translocates from the polymerase to the exonuclease site and the corrected DNA terminus returns has remained elusive. Here, we present an ensemble of nine high-resolution structures representing human mitochondrial DNA polymerase Gamma, Polγ, captured during consecutive proofreading steps. The structures reveal key events, including mismatched base recognition, its dissociation from the polymerase site, forward translocation of DNAP, alterations in DNA trajectory, repositioning and refolding of elements for primer separation, DNAP backtracking, and displacement of the mismatched base into the exonuclease site. Altogether, our findings suggest a conserved \u27bolt-action\u27 mechanism of proofreading based on iterative cycles of DNAP translocation without dissociation from the DNA, facilitating primer transfer between catalytic sites. Functional assays and mutagenesis corroborate this mechanism, connecting pathogenic mutations to crucial structural elements in proofreading steps

Mechanism of Transcription Initiation and Primer Generation at the Mitochondrial Origin of Replication, OriL

The intricate process of human mtDNA replication requires the coordinated action of both transcription and replication machineries. Transcription and replication events at the lagging strand of mtDNA prompt the formation of a stem-loop structure (OriL) and the synthesis of a ~25 nt RNA primer by mitochondrial RNA polymerase (mtRNAP). The mechanisms by which mtRNAP recognizes OriL, initiates transcription, and transfers the primer to the replisome are poorly understood. To address these mechanisms, we reconstituted OriL in vitro transcription and coupled transcription-replication assays. We found that unlike promoter-driven transcription, OriL transcription is independent of transcription factors and does not require the structural elements of mtRNAP involved in promoter recognition. However, the efficiency of transcription at OriL is significantly lower than the one at the light-strand promoter (LSP). We defined the OriL transcription start site and found that transcription initiation involves slippage of the nascent transcript, a process of repetitive addition of AMP residues to the transcript without translocation of polymerase relative to the DNA template. Our results indicate that the transcript slippage is required for initiation complex stability and its ability to generate RNA primer without translocation. Our data suggest a model of transcription replication, according to which the mitochondrial DNA polymerase gamma, PolG, pre-binds to the stem region of OriL, leaving the loop of the OriL hairpin available to mtRNAP binding. Upon generation of ~30 nt RNA primer, mtRNAP dissociates and the 3’ end of the RNA is taken over by PolG, which commences synthesis of the nascent DNA strand. Our data suggest the primosome assembly at OriL - a complex of mtRNAP and PolG - can efficiently generate the primer, transfer it to the replisome, and protect it from degradation by mitochondrial endonucleases

Structural basis for DNA proofreading

Abstract DNA polymerase (DNAP) can correct errors in DNA during replication by proofreading, a process critical for cell viability. However, the mechanism by which an erroneously incorporated base translocates from the polymerase to the exonuclease site and the corrected DNA terminus returns has remained elusive. Here, we present an ensemble of nine high-resolution structures representing human mitochondrial DNA polymerase Gamma, Polγ, captured during consecutive proofreading steps. The structures reveal key events, including mismatched base recognition, its dissociation from the polymerase site, forward translocation of DNAP, alterations in DNA trajectory, repositioning and refolding of elements for primer separation, DNAP backtracking, and displacement of the mismatched base into the exonuclease site. Altogether, our findings suggest a conserved ‘bolt-action’ mechanism of proofreading based on iterative cycles of DNAP translocation without dissociation from the DNA, facilitating primer transfer between catalytic sites. Functional assays and mutagenesis corroborate this mechanism, connecting pathogenic mutations to crucial structural elements in proofreading steps