Abstract
Solution nuclear magnetic resonance (NMR) experiments allow RNA dynamics to be determined in an aqueous environment. However, when a limited number of peaks are assigned, it is difficult to obtain structural information. We here show a protocol based on the combination of experimental data (Nuclear Overhauser Effect, NOE) and molecular dynamics simulations with enhanced sampling methods. This protocol allows to (a) obtain a maximum entropy ensemble compatible with NMR restraints and (b) obtain a minimal set of metastable conformations compatible with the experimental data (maximum parsimony). The method is applied to a hairpin of 29 nt from an inverted SINEB2, which is part of the SINEUP family and has been shown to enhance protein translation. A clustering procedure is introduced where the annotation of base-base interactions and glycosidic bond angles is used as a metric. By reweighting the contributions of the clusters, minimal sets of four conformations could be found which are compatible with the experimental data. A motif search on the structural database showed that some identified low-population states are present in experimental structures of other RNA transcripts. The introduced method can be applied to characterize RNA dynamics in systems where a limited amount of NMR information is available.
Highlights
RNA plays a fundamental role in the cell
We here show a protocol based on the combination of experimental data (Nuclear Overhauser Effect, NOE) and molecular dynamics simulations with enhanced sampling methods
We showed how enhanced sampling techniques in Molecular dynamics (MD) simulations can be synergistically combined with nuclear magnetic resonance (NMR) experimental restraints to obtain such a diverse ensemble and how results can be analyzed so as to be interpreted in terms of a reduced number of molecular conformations
Summary
RNA plays a fundamental role in the cell. It encodes the amino acid sequence of proteins (messenger RNA, mRNA) [1], is used as an adapter in translation (transfer RNA, tRNA) [2] and performs protein synthesis (ribosomal RNA, rRNA) [3]. In the last decades a growing number of non-coding RNAs have been discovered playing important roles in regulation [4,5]. RNA function is often linked to its conformational dynamics rather than to a unique structure [6,7]. Extreme examples in this sense are riboswitches [8], that can adopt different, competing metastable structures whose relative stability is controlled by the cellular environment. Advanced nuclear magnetic resonance (NMR) techniques, and in particular relaxation dispersion methods [9], provide a powerful approach to the identification of so-called ‘excited states’ in solution and have been used to identify transient states in RNA [10]
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