tRNA Identity Mediated Control of the Catalytic mechanism in E. coli Histidyl-tRNA Synthetase

The aminoacyl-tRNA synthetases (aaRSs) are the universal set of enzymes responsible for attaching amino acids to tRNA to be used as substrates in the process of protein translation. As these enzymes act at the transition between nucleic acids and proteins, their specificity of action is critical for maintaining the fidelity of the genetic code. From a mechanistic standpoint, aaRS specificity is enforced by a complex series of tRNA structural and chemical elements that collectively make up its identity set and serve to distinguish one tRNA from another. Based on sequence, structure, and oligomeric differences, the aaRS family has been partitioned into two classes, each of which is responsible for roughly half of the 22 genetically encoded amino acids. In the studies presented here, pre-steady-state kinetic methods were employed to measure individual events that collectively make up the catalytic cycle of the class II Escherichia coli Histidyl-tRNA Synthetase (HisRS) in order to elucidate the nature of its enzymatic activity and determine how these events contribute to the exquisite specificity between enzyme and tRNA. The results presented here indicate indentiy elements of tRNAHis regulate the activity of the amino acid activation and aminoacyl transfer half reactions. Additional evidence suggests communication between active sites of the HisRS homodimer plays a role in establishing an alternating cycle of catalysis in the steady state

Study of the B−→Λc+Λˉc−K−B^{-} \to \Lambda_{c}^{+} \bar{\Lambda}_{c}^{-} K^{-} decay

The decay $B^{-} \to \Lambda_{c}^{+} \bar{\Lambda}_{c}^{-} K^{-}$ is studied in proton-proton collisions at a center-of-mass energy of $\sqrt{s}=13$ TeV using data corresponding to an integrated luminosity of 5 $\mathrm{fb}^{-1}$ collected by the LHCb experiment. In the $\Lambda_{c}^+ K^{-}$ system, the $\Xi_{c}(2930)^{0}$ state observed at the BaBar and Belle experiments is resolved into two narrower states, $\Xi_{c}(2923)^{0}$ and $\Xi_{c}(2939)^{0}$, whose masses and widths are measured to be $$m(\Xi_{c}(2923)^{0}) = 2924.5 \pm 0.4 \pm 1.1 \,\mathrm{MeV}, \\ m(\Xi_{c}(2939)^{0}) = 2938.5 \pm 0.9 \pm 2.3 \,\mathrm{MeV}, \\ \Gamma(\Xi_{c}(2923)^{0}) = \phantom{000}4.8 \pm 0.9 \pm 1.5 \,\mathrm{MeV},\\ \Gamma(\Xi_{c}(2939)^{0}) = \phantom{00}11.0 \pm 1.9 \pm 7.5 \,\mathrm{MeV},$$ where the first uncertainties are statistical and the second systematic. The results are consistent with a previous LHCb measurement using a prompt $\Lambda_{c}^{+} K^{-}$ sample. Evidence of a new $\Xi_{c}(2880)^{0}$ state is found with a local significance of $3.8\,\sigma$, whose mass and width are measured to be $2881.8 \pm 3.1 \pm 8.5\,\mathrm{MeV}$ and $12.4 \pm 5.3 \pm 5.8 \,\mathrm{MeV}$, respectively. In addition, evidence of a new decay mode $\Xi_{c}(2790)^{0} \to \Lambda_{c}^{+} K^{-}$ is found with a significance of $3.7\,\sigma$. The relative branching fraction of $B^{-} \to \Lambda_{c}^{+} \bar{\Lambda}_{c}^{-} K^{-}$ with respect to the $B^{-} \to D^{+} D^{-} K^{-}$ decay is measured to be $2.36 \pm 0.11 \pm 0.22 \pm 0.25$, where the first uncertainty is statistical, the second systematic and the third originates from the branching fractions of charm hadron decays.Comment: All figures and tables, along with any supplementary material and additional information, are available at https://cern.ch/lhcbproject/Publications/p/LHCb-PAPER-2022-028.html (LHCb public pages

Measurement of the ratios of branching fractions R(D∗)\mathcal{R}(D^{*}) and R(D0)\mathcal{R}(D^{0})

The ratios of branching fractions $\mathcal{R}(D^{*})\equiv\mathcal{B}(\bar{B}\to D^{*}\tau^{-}\bar{\nu}_{\tau})/\mathcal{B}(\bar{B}\to D^{*}\mu^{-}\bar{\nu}_{\mu})$ and $\mathcal{R}(D^{0})\equiv\mathcal{B}(B^{-}\to D^{0}\tau^{-}\bar{\nu}_{\tau})/\mathcal{B}(B^{-}\to D^{0}\mu^{-}\bar{\nu}_{\mu})$ are measured, assuming isospin symmetry, using a sample of proton-proton collision data corresponding to 3.0 fb${ }^{-1}$ of integrated luminosity recorded by the LHCb experiment during 2011 and 2012. The tau lepton is identified in the decay mode $\tau^{-}\to\mu^{-}\nu_{\tau}\bar{\nu}_{\mu}$. The measured values are $\mathcal{R}(D^{*})=0.281\pm0.018\pm0.024$ and $\mathcal{R}(D^{0})=0.441\pm0.060\pm0.066$, where the first uncertainty is statistical and the second is systematic. The correlation between these measurements is $\rho=-0.43$. Results are consistent with the current average of these quantities and are at a combined 1.9 standard deviations from the predictions based on lepton flavor universality in the Standard Model.Comment: All figures and tables, along with any supplementary material and additional information, are available at https://cern.ch/lhcbproject/Publications/p/LHCb-PAPER-2022-039.html (LHCb public pages

Mycobacterial Ubiquitin-like Protein Ligase PafA Follows a Two-step Reaction Pathway with a Phosphorylated Pup Intermediate*

In Mycobacterium tuberculosis, the enzyme PafA is responsible for the activation and conjugation of the proteasome-targeting molecule Pup to protein substrates. As the proteasomal pathway has been shown to be vital to the persistence of M. tuberculosis, understanding the reaction mechanism of PafA is critical to the design of antituberculous agents. In this study, we have developed novel techniques to study the activity of PafA and have characterized fundamental features of the reaction mechanism. We show that PafA catalyzes a two-step reaction mechanism proceeding through a γ-glutamyl phosphate-mixed anhydride intermediate that is formed on the C-terminal glutamate of Pup before transfer of Pup to the substrate acceptor lysine. SDS-PAGE analysis of formation of the phosphorylated intermediate revealed that the rate of Pup activation matched the maximal steady-state rate of product formation in the overall reaction and suggested that Pup activation was rate-limiting when all substrates were present at saturating concentrations. Following activation, both ADP and the phosphorylated intermediate remained associated with the enzyme awaiting nucleophilic attack by a lysine residue of the target protein. The PafA reaction mechanism appeared to be noticeably biased toward the stable activation of Pup in the absence of additional substrate and required very low concentrations of ATP and Pup relative to other carboxylate-amine/ammonia ligase family members. The bona fide nucleophilic substrate PanB showed a 3 orders of magnitude stronger affinity than free lysine, promoting Pup conjugation to occur close to the rate limit of activation with physiologically relevant concentrations of substrate

Discovering and forecasting extreme events via active learning in neural operators

Extreme events in society and nature, such as pandemic spikes, rogue waves or structural failures, can have catastrophic consequences. Characterizing extremes is difficult, as they occur rarely, arise from seemingly benign conditions, and belong to complex and often unknown infinite-dimensional systems. Such challenges render attempts at characterizing them moot. We address each of these difficulties by combining output-weighted training schemes in Bayesian experimental design (BED) with an ensemble of deep neural operators. This model-agnostic framework pairs a BED scheme that actively selects data for quantifying extreme events with an ensemble of deep neural operators that approximate infinite-dimensional nonlinear operators. We show that not only does this framework outperform Gaussian processes, but that (1) shallow ensembles of just two members perform best; (2) extremes are uncovered regardless of the state of the initial data (that is, with or without extremes); (3) our method eliminates 'double-descent' phenomena; (4) the use of batches of suboptimal acquisition samples compared to step-by-step global optima does not hinder BED performance; and (5) Monte Carlo acquisition outperforms standard optimizers in high dimensions. Together, these conclusions form a scalable artificial intelligence (AI)-assisted experimental infrastructure that can efficiently infer and pinpoint critical situations across many domains, from physical to societal systems

Evolutionary conservation of a functionally important backbone phosphate group critical for aminoacylation of histidine tRNAs

All histidine tRNA molecules have an extra nucleotide, G-1, at the 5′ end of the acceptor stem. In bacteria, archaea, and eukaryotic organelles, G-1 base pairs with C73, while in eukaryotic cytoplasmic tRNA(His), G-1 is opposite A73. Previous studies of Escherichia coli histidyl-tRNA synthetase (HisRS) have demonstrated the importance of the G-1:C73 base pair to tRNA(His) identity. Specifically, the 5′-monophosphate of G-1 and the major groove amine of C73 are recognized by E. coli HisRS; these individual atomic groups each contribute ∼4 kcal/mol to transition state stabilization. In this study, two chemically synthesized 24-nucleotide RNA microhelices, each of which recapitulates the acceptor stem of either E. coli or Saccharomyces cervisiae tRNA(His), were used to facilitate an atomic group “mutagenesis” study of the −1:73 base pair recognition by S. cerevisiae HisRS. Compared with E. coli HisRS, microhelix(His) is a much poorer substrate relative to full-length tRNA(His) for the yeast enzyme. However, the data presented here suggest that, similar to the E. coli system, the 5′ monophosphate of yeast tRNA(His) is critical for aminoacylation by yeast HisRS and contributes ∼3 kcal/mol to transition state stability. The primary role of the unique −1:73 base pair of yeast tRNA(His) appears to be to properly position the critical 5′ monophosphate for interaction with the yeast enzyme. Our data also suggest that the eukaryotic HisRS/tRNA(His) interaction has coevolved to rely less on specific major groove interactions with base atomic groups than the bacterial system

The Usher Syndrome Type IIIB Histidyl-tRNA Synthetase Mutation Confers Temperature Sensitivity

Histidyl-tRNA synthetase (HARS) is a highly conserved translation factor that plays an essential role in protein synthesis. HARS has been implicated in the human syndromes Charcot-Marie-Tooth (CMT) Type 2W and Type IIIB Usher (USH3B). The USH3B mutation, which encodes an Y454S substitution in HARS, is inherited in an autosomal recessive fashion and associated with childhood deafness, blindness and episodic hallucinations during acute illness. The biochemical basis of the pathophysiologies linked to USH3B is currently unknown. Here, we present a detailed functional comparison of wild type and Y454S HARS enzymes. Kinetic parameters for enzymes and canonical substrates were determined using both steady state and rapid kinetics. Enzyme stability was examined using differential scanning fluorimetry. Finally, enzyme functionality in primary cell culture was assessed. Our results demonstrate that the Y454S substitution leaves HARS amino acid activation, aminoacylation and tRNA(His) binding functions largely intact compared with WT HARS, and the mutant enzyme dimerizes similarly to wild type. Interestingly, during our investigation it was revealed that the kinetics of amino acid activation differs from the previously characterized bacterial HisRS. Despite the similar kinetics, differential scanning fluorimetry revealed that Y454S is less thermally stable than WT HARS, and cells from Y454S patients grown at elevated temperature demonstrate diminished protein synthesis compared to wild type cells. The thermal sensitivity associated with the Y454S mutation represents offers a biochemical basis for understanding USH3B