Abstract
RNA not only translates the genetic code into proteins, but also carries out important cellular functions. Understanding such functions requires knowledge of the structure and dynamics at atomic resolution. Almost half of the published RNA structures have been solved by nuclear magnetic resonance (NMR). However, as a result of severe resonance overlap and low proton density, high-resolution RNA structures are rarely obtained from nuclear Overhauser enhancement (NOE) data alone. Instead, additional semi-empirical restraints and labor-intensive techniques are required for structural averages, while there are only a few experimentally derived ensembles representing dynamics. Here we show that our exact NOE (eNOE) based structure determination protocol is able to define a 14-mer UUCG tetraloop structure at high resolution without other restraints. Additionally, we use eNOEs to calculate a two-state structure, which samples its conformational space. The protocol may open an avenue to obtain high-resolution structures of small RNA of unprecedented accuracy with moderate experimental efforts.
Highlights
RNA translates the genetic code into proteins, and carries out important cellular functions
The NOE spectroscopy (NOESY) acquired from the sample in H2O yielded 91 exact NOE (eNOE) involving exchangeable amino resonances, as well as the stable hydroxyl resonance of U6
The Watergate suppression of the water signal in the H2O NOESY was sub-par, a NOESY with water presaturation was acquired in D2O, which yielded 174 eNOEs between non-exchangeable base and ribose resonances
Summary
RNA translates the genetic code into proteins, and carries out important cellular functions. As a result of severe resonance overlap and low proton density, high-resolution RNA structures are rarely obtained from nuclear Overhauser enhancement (NOE) data alone. X-ray crystallography and cryo-electron microscopy (cryo-EM) are still the techniques of choice for investigating RNAs and RNA–protein complexes larger than 50 kDa, which are difficult to study using NMR due to spectral overlap and fast T2 relaxation times, recent methodological advances have made NMR an alternative for studies of RNA of such sizes[7,8,9,10,11] Despite these advantages, NMR has room for substantial improvement. Pioneering work on the extraction of exact distances in biomacromolecules from NOE buildup measurements was carried out on RNA26 as a Conventional NOE r
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