Human mitochondrial RNA polymerase structures reveal transcription start-site and slippage mechanism.

Human mitochondrial RNA polymerase (POLRMT) and protein factors TFAM and TFB2M assemble on mitochondrial DNA promoters to initiate promoter-specific transcription. We present cryo-EM structures of two initiation complexes, IC3 and slipped-IC3, with fully resolved transcription bubbles containing RNA transcripts starting from +1 and -1 positions, respectively. These structures reveal the mechanisms of promoter melting, start site selection, and slippage synthesis.

DNA Polymerase Locks Replication Fork Under Stress.

Replication of DNA requires the parental DNA to be unwound to allow the genetic information to be faithfully duplicated by the replisome. While this function is usually shared by a host of proteins in the replisome, notably DNA polymerase (DNAP) and helicase, the consequence of DNAP synthesizing DNA while decoupled from helicase remains not well understood. The unwinding of downstream DNA poses significant stress to DNAP, and the interaction between DNAP and the replication fork may affect replication restart.

Sequence-specific dynamic DNA bending explains mitochondrial TFAM's dual role in DNA packaging and transcription initiation.

Mitochondrial transcription factor A (TFAM) employs DNA bending to package mitochondrial DNA (mtDNA) into nucleoids and recruit mitochondrial RNA polymerase (POLRMT) at specific promoter sites, light strand promoter (LSP) and heavy strand promoter (HSP). Herein, we characterize the conformational dynamics of TFAM on promoter and non-promoter sequences using single-molecule fluorescence resonance energy transfer (smFRET) and single-molecule protein-induced fluorescence enhancement (smPIFE) methods. The DNA-TFAM complexes dynamically transition between partially and fully bent DNA conformational states.

Proofreading mechanisms of the innate immune receptor RIG-I: distinguishing self and viral RNA.

The RIG-I-like receptors (RLRs), comprising retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2), are pattern recognition receptors belonging to the DExD/H-box RNA helicase family of proteins. RLRs detect viral RNAs in the cytoplasm and respond by initiating a robust antiviral response that up-regulates interferon and cytokine production. RIG-I and MDA5 complement each other by recognizing different RNA features, and LGP2 regulates their activation.

The NS1 protein of influenza B virus binds 5'-triphosphorylated dsRNA to suppress RIG-I activation and the host antiviral response.

Influenza A and B viruses overcome the host antiviral response to cause a contagious and often severe human respiratory disease. Here, integrative structural biology and biochemistry studies on non-structural protein 1 of influenza B virus (NS1B) reveal a previously unrecognized viral mechanism for innate immune evasion. Conserved basic groups of its C-terminal domain (NS1B-CTD) bind 5'triphosphorylated double-stranded RNA (5'-ppp-dsRNA), the primary pathogen-associated feature that activates the host retinoic acid-inducible gene I protein (RIG-I) to initiate interferon synthesis and the cellular antiviral response.

Unraveling blunt-end RNA binding and ATPase-driven translocation activities of the RIG-I family helicase LGP2.

The RIG-I family helicases, comprising RIG-I, MDA5 and LGP2, are cytoplasmic RNA sensors that trigger an antiviral immune response by specifically recognizing foreign RNAs. While LGP2 lacks the signaling domain necessary for immune activation, it plays a vital role in regulating the RIG-I/MDA5 signaling pathway. In this study, we investigate the mechanisms underlying this regulation by examining the oligomeric state, RNA binding specificity, and translocation activity of human LGP2 and the impact of ATPase activity.

Twinkle-Catalyzed Toehold-Mediated DNA Strand Displacement Reaction.

Strand exchange between homologous nucleic acid sequences is the basis for cellular DNA repair, recombination, and genome editing technologies. Specialized enzymes catalyze cellular strand exchange; however, the reaction occurs spontaneously when a single-stranded DNA toehold can dock the invader strand on the target DNA to initiate strand exchange through branch migration. Due to its precise response, the spontaneous toehold-mediated strand displacement (TMSD) reaction is widely employed in DNA nanotechnology.

Structures illustrate step-by-step mitochondrial transcription initiation.

Transcription initiation is a key regulatory step in gene expression during which RNA polymerase (RNAP) initiates RNA synthesis de novo, and the synthesized RNA at a specific length triggers the transition to the elongation phase. Mitochondria recruit a single-subunit RNAP and one or two auxiliary factors to initiate transcription. Previous studies have revealed the molecular architectures of yeast and human mitochondrial RNAP initiation complexes (ICs).

RIG-I recognizes metabolite-capped RNAs as signaling ligands.

The innate immune receptor RIG-I recognizes 5'-triphosphate double-stranded RNAs (5' PPP dsRNA) as pathogenic RNAs. Such RNA-ends are present in viral genomes and replication intermediates, and they activate the RIG-I signaling pathway to produce a potent interferon response essential for viral clearance. Endogenous mRNAs cap the 5' PPP-end with m7G and methylate the 2'-O-ribose to evade RIG-I, preventing aberrant immune responses deleterious to the cell.

The N-terminal domain of human mitochondrial helicase Twinkle has DNA-binding activity crucial for supporting processive DNA synthesis by polymerase γ.

Twinkle is the ring-shaped replicative helicase within the human mitochondria with high homology to bacteriophage T7 gp4 helicase-primase. Unlike many orthologs of Twinkle, the N-terminal domain (NTD) of human Twinkle has lost its primase activity through evolutionarily acquired mutations. The NTD has no demonstrated activity thus far; its role has remained unclear.

Quantitative methods to study helicase, DNA polymerase, and exonuclease coupling during DNA replication.

Genome replication is accomplished by highly regulated activities of enzymes in a multi-protein complex called the replisome. Two major enzymes, DNA polymerase and helicase, catalyze continuous DNA synthesis on the leading strand of the parental DNA duplex while the lagging strand is synthesized discontinuously. The helicase and DNA polymerase on their own are catalytically inefficient and weak motors for unwinding/replicating double-stranded DNA.

The intrinsically disordered CARDs-Helicase linker in RIG-I is a molecular gate for RNA proofreading.

The innate immune receptor RIG-I provides a first line of defense against viral infections. Viral RNAs are recognized by RIG-I's C-terminal domain (CTD), but the RNA must engage the helicase domain to release the signaling CARD (Caspase Activation and Recruitment Domain) domains from their autoinhibitory CARD2:Hel2i interactions. Because the helicase itself lacks RNA specificity, mechanisms to proofread RNAs entering the helicase domain must exist.

Assembly and Cryo-EM structure determination of yeast mitochondrial RNA polymerase initiation complex intermediates.

In yeast mitochondria, transcription initiation requires assembly of mitochondrial RNA polymerase and transcription initiation factor MTF1 at the DNA promoter initiation site. This protocol describes the purification of the component proteins and assembly of partially melted and fully melted initiation complex states. Both states co-exist in equilibrium in the same sample as seen by cryoelectron microscopy (cryo-EM) and allow elucidation of MTF1's structural roles in controlling the transition into elongation.

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.

Structure, mechanism, and regulation of mitochondrial DNA transcription initiation.

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 dynamic landscape of transcription initiation in yeast mitochondria.

Controlling efficiency and fidelity in the early stage of mitochondrial DNA transcription is crucial for regulating cellular energy metabolism. Conformational transitions of the transcription initiation complex must be central for such control, but how the conformational dynamics progress throughout transcription initiation remains unknown. Here, we use single-molecule fluorescence resonance energy transfer techniques to examine the conformational dynamics of the transcriptional system of yeast mitochondria with single-base resolution.

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).

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 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.

Excessive excision of correct nucleotides during DNA synthesis explained by replication hurdles.

The proofreading exonuclease activity of replicative DNA polymerase excises misincorporated nucleotides during DNA synthesis, but these events are rare. Therefore, we were surprised to find that T7 replisome excised nearly 7% of correctly incorporated nucleotides during leading and lagging strand syntheses. Similar observations with two other DNA polymerases establish its generality.

The C-terminal tail of the yeast mitochondrial transcription factor Mtf1 coordinates template strand alignment, DNA scrunching and timely transition into elongation.

Mitochondrial RNA polymerases depend on initiation factors, such as TFB2M in humans and Mtf1 in yeast Saccharomyces cerevisiae, for promoter-specific transcription. These factors drive the melting of promoter DNA, but how they support RNA priming and growth was not understood. We show that the flexible C-terminal tails of Mtf1 and TFB2M play a crucial role in RNA priming by aiding template strand alignment in the active site for high-affinity binding of the initiating nucleotides.