Biology subjects

York, D. M.

Publications and source records attributed to York, D. M..

4 recordsLinked to original sources

bioRxiv2026

A transient third metal ion shapes the catalytic cycle of a group I intron ribozyme

The self-splicing group I intron ribozyme from Tetrahymena thermophillia (THr) is a prototypical example of how RNA overcomes its limited chemical repertoire to form complex, negatively charged junctions and catalyze chemical reactions. Despite over 30 years of biochemical and structural characterization, questions still remain regarding the requirements for THr catalysis that may inform structure based drug design against group I introns, as well as the use of THr as an RNA editing tool. Recent cryo-EM structures have provided an unprecedented window into the first and second steps of splicing, enabling detailed mechanistic investigation at the atomistic level. In this work, we leverage solvation theory, molecular dynamics, alchemical free energy simulations, and machine learning accelerated QM/MM to probe outstanding questions related to 1) the role of a possible third Mg2+ binding in the active site, 2) differences in catalytic requirements between Gexo cofactors and between the first and second steps of splicing, 3) the potential role of an acid or base, and 4) restrictions on the identity of the 5' exon splice site nucleotide, U(-1). We propose that a transient, cofactor dependent, interaction with a third metal participates in the first step, while a higher affinity interaction with this third ion is required in the second step. We also propose that the presence of a phosphate on the Gexo in the first step, and analogously the G{omega} in the second step, forms a metal ion binding site not present when guanosine is bound. Finally, we propose and computationally test design modifications of G22 (isofunctional substitutions of 2,6-diaminopurine that tune the pKa at the N1 position) that we predict will enable the accommodation of C(-1) in a crucial wobble-like pair at physiological pH. This design would provide a potential strategy for expanding the sequence space accessible to programmable trans-splicing ribozymes in RNA editing applications.

biophysics↗

bioRxiv2026

Origins of reactivity in SAM-utilizing ribozyme SAMURI-catalyzed RNA alkylation

Unlocking the design principles of programmable RNA catalysts capable of sitespecific chemical modification is critical for expanding the functional and therapeutic potential of RNA. The SAM analogue-utilizing ribozyme (SAMURI) enables sitespecific RNA alkylation using either S-adenosylmethionine (SAM) or the synthetic cofactor propargylic Se-2,6-diaminopurinribosyl-selenomethionineamide (ProSeDMA), yet the molecular determinants of its reactivity remain incompletely understood. Here, we combined molecular dynamics, 3D-RISM solvation analysis, alchemical free-energy calculations, quantum pKa shift predictions, and ab initio QM/MM free-energy simulations to characterize the conformational and electronic factors that govern catalysis. Simulations show that, although the global fold of SAMURI remains stable in solution, the formation of catalytically competent near-attack configurations is rare, indicating that the observed rate depends on access to a minor fraction of these reactive conformations (freact). A putative Mg2+ binding site between the SAM carboxylate and the G30 phosphate, together with a hydrogen bond between the cofactor -amine and U8:O2, enriches freact. QM/MM simulations support an SN2-like alkyl transfer mechanism and show that ProSeDMA reacts more readily than SAM primarily due to its more favorable electronic leaving-group properties that enhance the intrinsic rate (kint). Atomic substitutions at A52 that tune the N3 pKa enhance nucleophilicity, further lower the activation barrier, and increase kint. Together, these results show that SAMURI catalysis is governed by a combination of conformational preorganization and electronic effects, providing a framework to guide the design of new programmable RNA alkyltransferases. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/720726v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@90e0c1org.highwire.dtl.DTLVardef@193b8d6org.highwire.dtl.DTLVardef@14c0d49org.highwire.dtl.DTLVardef@1fc6793_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Structural Plasticity of RRE Stem-Loop II Modulates Nuclear Export of HIV-1 RNA

The Rev Response Element (RRE) forms an oligomeric complex with the viral protein Rev to facilitate the nuclear export of intron-retaining viral RNAs during the late phase of HIV-1 infection. However, our structural understanding of this crucial virological process remains limited. In this study, we determined several crystal structures of an intact RRE stem-loop II in two distinct conformations, performed negative-staining electron microscopy and molecular dynamics simulations, and revealed that this three-way junction RNA exhibits remarkable structural plasticity. Through in vitro Rev-binding and in vivo Rev-activity assays using various stem-loop II mutants designed to favor one of the conformers, we demonstrated that the structural plasticity of stem-loop II modulates Rev binding and oligomerization. Our findings illuminate emerging perspectives on RRE dynamics-based regulation of HIV-1 RNA nuclear export and provide a framework for developing anti-HIV drugs that target specific RRE conformations.

biophysics↗

bioRxiv2021

Introducing a New Bond-Forming Activity in an Archaeal DNA Polymerase by Structure-Guided Enzyme Redesign

DNA polymerases have evolved to feature a highly conserved activity across the tree of life: formation of, without exception, phosphodiester linkages that create the repeating sugarphosphate backbone of DNA. Can this linkage selectivity observed in nature be overcome by design to produce non-natural nucleic acids? Here, we report that structure-guided redesign of an archaeal DNA polymerase (9{degrees}N) enables a new polymerase activity that is undetectable in the wild type enzyme: catalyzing the formation of N3[->]P5 phosphoramidate linkages in the presence of 3-amino-2,3-dideoxynucleoside 5-triphosphate (3-NH2-ddNTP) building blocks. Replacing a highly conserved metal-binding aspartate in the 9{degrees}N active site (Asp-404) with asparagine was key to the emergence of this unnatural enzyme activity. Molecular dynamics simulations provided insights into how a single substitution could enhance the productive positioning of the 3-amino nucleophile in the active site. Further remodeling of the protein-nucleic acid interface with substitutions in the finger subdomain led to a quadruple-mutant variant (9{degrees}N-NRQS) that incorporated 3-NH2-ddNTPs into a 3-amino-primer on various DNA templates. This work presents the first example of an active-site substitution of a metal-binding residue that leads to a novel activity in a DNA polymerase, and sheds light on the molecular basis of substrate fidelity and latent promiscuity in enzymes.

biochemistry↗