Biology subjects

Mishanina, T. V.

Publications and source records attributed to Mishanina, T. V..

8 recordsLinked to original sources

bioRxiv2026

Pathogenic human mitochondrial tRNA variants impair RNA processing by compromising 5' leader removal

Human mitochondrial genome (mtDNA) encodes multiple proteins in the oxidative phosphorylation complexes as well as the ribosomal and transfer RNAs (tRNAs) needed for in situ translation. These genes are transcribed from only three promoters, producing polycistronic transcripts that are co-transcriptionally cleaved by mitochondrial RNase enzymes to release majority of individual gene products. tRNAs separate many of these genes and are thought to serve as "punctuation" marks that enable RNase recognition, binding, and hydrolysis of the 5' "leader" and 3' "trailer" sequences flanking the tRNA. Mutations in the tRNA genes dominate the mtDNA-linked mitochondrial pathologies; yet a systematic study of the impact of tRNA sequence variation on the RNase-catalyzed processing is lacking. Here, we employed human mitochondrial tRNATyr as a model system to dissect the effect of tRNA variants on the in vitro 5' leader and 3' trailer hydrolysis. We found that nucleotide variations located near the catalytic interfaces - particularly within or near the tRNA acceptor stem - showed the strongest defects in 5' processing and prevented release of the downstream tRNA in a tRNA cluster where multiple tRNAs are transcribed in tandem. This work provides mechanistic insight into how mutations disrupt coordinated mitochondrial tRNA processing and establish a framework for predicting variant effects based on their structural position relative to the processing enzymes.

biochemistry↗

bioRxiv2026

pH-dependent allosteric remodeling of a bacterial riboswitch couples alkaline activation to metal sensing

The widespread yybP-ykoY riboswitches control bacterial manganese (Mn) homeostasis by activating exporter expression in response to intracellular Mn2+ levels. The E. coli alx riboswitch distinctively couples Mn2+ sensing to cytoplasmic alkalinity, but the mechanism is unknown. We show that pH tunes the alx aptamers conformational sampling to modulate Mn2+ sensitivity. Single-molecule FRET reveals that Mn2+ stabilizes a docked three-way-junction conformation, and alkaline pH shifts this equilibrium to sensitize metal-dependent folding. Molecular dynamics simulations identify a loop whose low-pH-induced base pairing perturbs the adjacent helix, predicted to allosterically disrupt the Mn2+-binding state. In vivo reporters indicate that both this loop and the Mn2+-binding core are required for optimal pH-dependent translational activation: replacing the core with the non-pH-responsive mntP sequence abolishes activation. These results define how RNA allosterically integrates orthogonal metal and proton cues to enable combinatorial environmental sensing during alkaline stress.

biochemistry↗

bioRxiv2025

Cellular metabolic state controls mitochondrial RNA kinetics

Human mitochondrial genome encodes essential genes for the oxidative phosphorylation (OXPHOS) complexes. These genes must be transcribed and translated in coordination with nuclear-encoded OXPHOS components to ensure correct stoichiometry during OXPHOS complex assembly in the mitochondria. While much is known about nuclear gene regulation during metabolic stresses like glucose deprivation, little is known about the accompanying transcriptional response in mitochondria. Using microscopy, roadblocking qPCR, and transcriptomics, we studied mitochondrial transcription in cells subjected to glucose deprivation, which is known to cause nuclear transcription downregulation and to activate the integrated stress response (ISR). We found that glucose deprivation stabilizes mitochondrial RNAs and slows mitochondrial transcription, effects that are quickly reversed with glucose reintroduction. Although transcriptomics revealed strong upregulation of the ISR, mitochondrial RNA stabilization was not upregulated by pharmacological activation of the ISR, but was promoted by inhibition of glycolysis, unveiling a direct connection between metabolism and regulation of mitochondrial gene expression.

molecular biology↗

bioRxiv2025

Nucleic acid sequence determinants oftranscriptional pausing by human mitochondrialRNA polymerase (POLRMT)

Transcription by RNA polymerase (RNAP) lies at the heart of gene expression in all organisms. The speed with which RNAPs produce the RNA is tuned in part by the signals in the transcribed nucleic-acid sequences, which temporarily arrange RNAPs into a paused conformation unable to extend the RNA. In turn, the altered transcription kinetics determines the three-dimensional shape into which RNA ultimately folds, dictates the chromatin state, and promotes or inhibits co-transcriptional events. While pause sequence determinants have been characterized for multi-subunit RNAPs in bacteria and the eukaryotic nuclei, this information is lacking for the single-subunit RNAP of human mitochondria, POLRMT. Here, we developed a robust nucleic-acid scaffold system to reconstitute POLRMT transcription in vitro and identified multiple transcriptional pause sites on the human mitochondrial genomic sequence (mtDNA). Using one of the pause sequences as a representative, we performed a suite of mutational studies to pinpoint the nucleic-acid elements that enhance, weaken, or completely abolish POLRMT pausing. Finally, a search of the human mtDNA for the pause motif revealed multiple predicted pause sites, with potential roles in mitochondrial co-transcriptional processes.

biochemistry↗

bioRxiv2023

A riboswitch-controlled manganese exporter (Alx) tunes intracellular Mn2+ concentration in E. coli at alkaline pH

Cells use transition metal ions as structural components of biomolecules and cofactors in enzymatic reactions, making transition metals vital cellular components. The buildup of a particular metal ion in certain stress conditions becomes harmful to the organism due to the misincorporation of the excess ion into biomolecules, resulting in perturbed enzymatic activity or metal-catalyzed formation of reactive oxygen species. Organisms optimize metal concentration by regulating the expression of proteins that import and export that metal, often in a metal concentration-dependent manner. One such regulation mechanism is via riboswitches, which are 5-untranslated regions (UTR) of an mRNA that undergo conformational changes to promote or inhibit the expression of the downstream gene, commonly in response to a ligand. The yybP-ykoY family of bacterial riboswitches shares a conserved aptamer domain that binds manganese (Mn2+). In E. coli, the yybP-ykoY riboswitch precedes and regulates the expression of two genes: mntP, which based on extensive genetic evidence encodes an Mn2+ exporter, and alx, which encodes a putative metal ion transporter whose cognate ligand is currently in question. Expression of alx is upregulated by both elevated intracellular concentrations of Mn2+ and alkaline pH. With metal ion measurements and gene expression studies, we demonstrate that the alkalinization of media increases cytoplasmic Mn2+ content, which in turn enhances alx expression. Alx then exports excess Mn2+ to prevent toxic buildup of the metal inside the cell, with the export activity maximal at alkaline pH. Using mutational and complementation experiments, we pinpoint a set of acidic residues in the predicted transmembrane segments of Alx that play a crucial role in its Mn2+ export. We propose that Alx-mediated Mn2+ export provides a primary protective layer that fine-tunes the cytoplasmic Mn2+ levels, especially during alkaline stress.

microbiology↗

bioRxiv2022

An ensemble of interconverting conformations of the elemental paused transcription complex creates regulatory options

Transcriptional pausing underpins regulation of cellular RNA synthesis but its mechanism remains incompletely understood. Sequence-specific interactions of DNA and RNA with the dynamic, multidomain RNA polymerase (RNAP) trigger reversible conformational changes at pause sites that temporarily interrupt the nucleotide addition cycle. These interactions initially rearrange the elongation complex (EC) into an elemental paused EC (ePEC). ePECs can form longer-lived PECs by further rearrangements or interactions of diffusible regulators. For both bacterial and mammalian RNAPs, a half-translocated state in which the next DNA template base fails to load into the active site appears central to the ePEC. Some RNAPs also swivel interconnected modules that may stabilize the ePEC. However, it is unclear if swiveling and half-translocation are requisite features of a single ePEC state or if multiple ePEC states exist. Here we use cryo-EM analysis of ePECs with different RNA-DNA sequences combined with biochemical probes of ePEC structure to define an interconverting ensemble of ePEC states. ePECs occupy either pre- or half-translocated states but do not always swivel, indicating that difficulty in forming the post-translocated state at certain RNA-DNA sequences may be the essence of the ePEC. The existence of multiple ePEC conformations has broad implications for transcriptional regulation. SIGNIFICANCETranscriptional pausing provides a hub for gene regulation. Pausing provides a timing mechanism to coordinate regulatory interactions, co-transcriptional RNA folding and protein synthesis, and stop signals for transcriptional termination. Cellular RNA polymerases (RNAPs) are complex, with multiple mobile modules shifting positions to control its catalytic activity and pause RNAP in response to DNA-encoded pause signals. Understanding how these modules move to enable pausing is crucial for a mechanistic understanding of gene regulation. Our results clarify the picture significantly by defining multiple states among which paused RNAP partitions in response to different pause signals. This work contributes to an emerging theme wherein multiple interconverting states of the RNAP proceed through a pathway (e.g., initiation or pausing), providing multiple opportunities for regulation.

biochemistry↗

bioRxiv2018

The elemental mechanism of transcriptional pausing

Transcriptional pausing underpins regulation of cellular RNA biogenesis. A consensus pause sequence that acts on RNA polymerases (RNAPs) from bacteria to mammals halts RNAP in an elemental paused state from which longer-lived pauses can arise. Although the structural foundations of pauses prolonged by backtracking or nascent RNA hairpins are recognized, the fundamental mechanism of the elemental pause is less well-defined. Here we report a mechanistic dissection that establishes the elemental pause signal (i) is multipartite; (ii) causes a modest conformational shift that puts RNAP in an off-pathway state in which template base loading but not RNA translocation is inhibited; and (iii) can easily enter pretranslocated and one-base-pair backtracked states despite principally occupying the half-translocated state observed in cryo-EM structures of paused RNAPs. Our findings provide a mechanistic basis for the elemental pause and a framework to understand how pausing is modulated by sequence, cellular conditions, and regulators.

biochemistry↗

bioRxiv2018

Structural basis for transcript elongation control by NusG/RfaH universal regulators

NusG/RfaH/Spt5 transcription elongation factors are the only transcription regulators conserved across all life. In bacteria, NusG regulates RNA polymerase (RNAP) elongation complexes (ECs) across most genes, enhancing elongation by suppressing RNAP backtracking and also coordinating {rho}-dependent termination and translation. RfaH is a specialized NusG paralog that engages the EC at ops sites and subsequently excludes NusG and suppresses both backtrack and hairpin-stabilized pausing. We used single-particle cryo-EM to determine structures of ECs at ops with NusG or RfaH. Both factors chaperone base pairing of the EC upstream duplex DNA to suppress backtracking. RfaH loads onto the EC by specific recognition of an ops hairpin in the single-stranded nontemplate DNA. Binding of both NusG and RfaH is incompatible with the swiveled RNAP conformation necessary for hairpin-stabilized pausing, but only RfaH fully counteracts swiveling to suppress pausing. The universal conservation of NusG/RfaH/Spt5 suggests that the molecular mechanisms uncovered here are widespread.

biophysics↗