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Resolving sugar puckers in RNA excited states exposes slow modes of repuckering dynamics.

ABSTRACT: Recent studies have shown that RNAs exist in dynamic equilibrium with short-lived low-abundance 'excited states' that form by reshuffling base pairs in and around non-canonical motifs. These conformational states are proposed to be rich in non-canonical motifs and to play roles in the folding and regulatory functions of non-coding RNAs but their structure proves difficult to characterize given their transient nature. Here, we describe an approach for determining sugar pucker conformation in RNA excited states through nuclear magnetic resonance measurements of C1? and C4? rotating frame spin relaxation (R1?) in uniformly 13C/15N labeled RNA samples. Application to HIV-1 TAR exposed slow modes of sugar repuckering dynamics at the ?s and ms timescale accompanying transitions between non-helical (C2?-endo) to helical (C3?-endo) conformations during formation of two distinct excited states. In contrast, we did not obtain any evidence for slow sugar repuckering dynamics for nucleotides in a variety of structural contexts that do not undergo non-helical to helical transitions. Our results outline a route for significantly improving the conformational characterization of RNA excited states and suggest that slow modes of repuckering dynamics gated by transient changes in secondary structure are quite common in RNA.

SUBMITTER: Clay MC

PROVIDER: S-EPMC5737546 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Resolving sugar puckers in RNA excited states exposes slow modes of repuckering dynamics.

Clay Mary C MC Ganser Laura R LR Merriman Dawn K DK Al-Hashimi Hashim M HM

Nucleic acids research 20170801 14

Recent studies have shown that RNAs exist in dynamic equilibrium with short-lived low-abundance 'excited states' that form by reshuffling base pairs in and around non-canonical motifs. These conformational states are proposed to be rich in non-canonical motifs and to play roles in the folding and regulatory functions of non-coding RNAs but their structure proves difficult to characterize given their transient nature. Here, we describe an approach for determining sugar pucker conformation in RNA ...[more]

PMID: 28609788

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Project description:Cyclin-dependent kinases (CDKs) and their associated regulatory cyclins are central for timely regulation of cell-cycle progression. They constitute attractive pharmacological targets for development of anticancer therapeutics, since they are frequently deregulated in human cancers and contribute to sustained, uncontrolled tumor proliferation. Characterization of their structural/dynamic features is essential to gain in-depth insight into structure-activity relationships. In addition, the identification of druggable pockets or key intermediate conformations yields potential targets for the development of novel classes of inhibitors. Structural studies of CDK2/cyclin A have provided a wealth of information concerning monomeric/heterodimeric forms of this kinase. There is, however, much less structural information for other CDK/cyclin complexes, including CDK4/cyclin D1, which displays an alternative (open) position of the cyclin partner relative to CDK, contrasting with the closed CDK2/cyclin A conformation. In this study, we carried out normal-mode analysis and enhanced sampling simulations with our recently developed method, molecular dynamics with excited normal modes, to understand the conformational equilibrium on these complexes. Interestingly, the lowest-frequency normal mode computed for each complex described the transition between the open and closed conformations. Exploration of these motions with an explicit-solvent representation using molecular dynamics with excited normal modes confirmed that the closed conformation is the most stable for the CDK2/cyclin A complex, in agreement with their experimentally available structures. On the other hand, we clearly show that an open↔closed equilibrium may exist in CDK4/cyclin D1, with closed conformations resembling that captured for CDK2/cyclin A. Such conformational preferences may result from the distinct distributions of frustrated contacts in each complex. Using the same approach, the putative roles of the Thr(160) phosphoryl group and the T-loop conformation were investigated. These results provide a dynamic view of CDKs revealing intermediate conformations not yet characterized for CDK members other than CDK2, which will be useful for the design of inhibitors targeting critical conformational transitions.

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Resolving sugar puckers in RNA excited states exposes slow modes of repuckering dynamics.

Publications

Resolving sugar puckers in RNA excited states exposes slow modes of repuckering dynamics.

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