Protein-RNA Interfaces Probed by ~1H-Detected MAS Solid-State NMR Spectroscopy

摘要

In the past decades, solid-state nuclear magnetic resonance (NMR) spectroscopy has evolved to become an important tool, enabling structural investigation of crystalline and, most notably, noncrystalline biomolecular systems. So far, mostly protein samples have been studied in the solid state, while only few investigations of RNA or DNA have been reported.'33 However, many fundamental biochemical processes are carried out by protein-RNA complexes, the most prominent of which is the translation machinery. Prior to the assembly of the eukaryotic ribosome, which catalyzes translation of messenger RNA, the RNA of the ribosomal subunits becomes extensively modified. The modifications consist of the conversion of uridine to pseudouridine and the methyl-ation of nucleobases and 2'-O-ribose units, which cluster predominantly within functionally crucial regions. It has been suggested that the modifications play an important role in RNA folding.In eukaryotes, the site-specific methylation of 2'-O-ribose units is guided by small nucleolar RNAs (snoRNAs).These snoRNAs are defined by two conserved sequence elements, the C box (RUGAUG, where R is purine) and D box (CUGA).The snoRNAs are complexed with specific proteins and assemble to form ribonucleoprotein particles (snoRNPs). In archaea, the small ribonucleoprotein particle (sRNP) is composed of a boxC/D snoRNA-like RNA (sRNA) and three proteins, L7Ae, Nop56/58, and fibrillarin, of which fibrillarin is the methyltransferase. The assembly of archaeal sRNP is initiated by the binding of L7Ae to box C/D RNA. Upon binding, the sRNA folds into a K-turn motif.Herein we suggest a new approach to characterize protein-RNA interfaces, which is based on proton-detected magic-angle-spinning (MAS) solid-state NMR experiments, using protonated and deuterated samples.