Recent analysis of transcriptomes has revealed that RNAs perform a myriad of functions beyond encoding proteins. Critical to RNA function is its transport to unique subcellular locations. Despite the importance of RNA localization, it is still very challenging to study in an unbiased manner. We recently described the ability to tag RNA molecules within subcellular locations through spatially restricted nucleobase oxidation. Herein, we describe a dramatic improvement of this protocol through the localized oxidation and tagging of proteins. Isolation of RNA-protein complexes enabled the enrichment of challenging RNA targets on chromatin and presented a considerably optimized protocol for the analysis of RNA subcellular localization within living cells.

RNase H degrades the RNA moiety in DNA:RNA hybrid in a divalent metal ion dependent manner. It is essential to understand the role of metal ion in enzymatic mechanism. One of the key points in this study is how many metal ions are involved in the enzyme catalysis. Accordingly, either one-metal binding mechanism or two-metal binding mechanism is proposed. We have studied the thermodynamic properties of four metal ions (Mg(2+), Mn(2+), Ca(2+), and Ba(2+)) binding to Methanococcus jannaschii RNase HII using isothermal titration calorimetry. All of the four metal ions were found to bind Mj RNase HII with 1:1 stoichiometry in the absence of substrate. Together with enzymatic activity assay data, we propose that only one metal ion binding to the enzyme in catalytic process. We also studied the pH dependence of metal binding and enzyme activity and found that at pH 6.5, Mg(2+) did not bind to the enzyme without the substrate but still activated the enzyme to about 2% of its maximum activity (in 10 mM Mn(2+) at pH 8). This implies that the substrate may also be incorporated in metal ion binding and help to position the metal ion. To find which acidic residues correspond to metal ion binding, we also studied the binding thermodynamics and enzymatic activity assay of four mutants: D7N, E8Q, D112N, and D149N in the presence of Mn(2+). The thermodynamic parameters are least affected for the D149N mutant, which has a very low enzymatic activity. This indicates that Asp149 is essential for the enzymatic activity. On the basis of all these observations, we suggest a metal binding model in which D7, E8, and D112 bind the metal ion and D149 activates a water molecule to attack the P-O bond in the RNA chain of the substrate.

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To enable efficient energy conversion schemes for our society in the future, breakthroughs in precise thermodynamic and kinetic control of the underlying redox reactions are necessary. Hybrid bilayer membranes (HBMs), comprising a self-assembled monolayer (SAM) covered by a lipid membrane, have been developed recently to regulate the performance of HBM-embedded electrocatalysts. A major technological roadblock in HBM development is the inability to facilitate proton transfer under alkaline conditions where nonprecious metal (NPM) catalysts can rival the performance of their precious metal counterparts. Here, we synthesized proton carriers bearing nitrile groups found in protonophores. These bioinspired proton carriers can facilitate transmembrane proton delivery to an HBM-supported Cu oxygen reduction reaction (ORR) catalyst under alkaline conditions. Our stimuli-responsive proton regulators can turn on the activity of the ORR catalyst on-demand, thereby opening doors to investigate how proton transfer kinetics govern the performance of electrocatalysts for renewable energy conversion processes.