Analysis of RNA structure using small-angle X-ray scattering

Methods. 2017 Jan 15;113:46-55. doi: 10.1016/j.ymeth.2016.10.008. Epub 2016 Oct 21.

Abstract

In addition to their role in correctly attaching specific amino acids to cognate tRNAs, aminoacyl-tRNA synthetases (aaRS) have been found to possess many alternative functions and often bind to and act on other nucleic acids. In contrast to the well-defined 3D structure of tRNA, the structures of many of the other RNAs recognized by aaRSs have not been solved. Despite advances in the use of X-ray crystallography (XRC), nuclear magnetic resonance (NMR) spectroscopy and cryo-electron microscopy (cryo-EM) for structural characterization of biomolecules, significant challenges to solving RNA structures still exist. Recently, small-angle X-ray scattering (SAXS) has been increasingly employed to characterize the 3D structures of RNAs and RNA-protein complexes. SAXS is capable of providing low-resolution tertiary structure information under physiological conditions and with less intensive sample preparation and data analysis requirements than XRC, NMR and cryo-EM. In this article, we describe best practices involved in the process of RNA and RNA-protein sample preparation, SAXS data collection, data analysis, and structural model building.

Keywords: Aminoacyl-tRNA synthetase; Native PAGE; RNA structure; SAXS; Size-exclusion chromatography; tRNA-like structures.

MeSH terms

  • Amino Acids / metabolism
  • Amino Acyl-tRNA Synthetases / genetics
  • Amino Acyl-tRNA Synthetases / metabolism*
  • Chromatography, Gel
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism
  • Models, Molecular
  • Native Polyacrylamide Gel Electrophoresis
  • Plasmids / chemistry
  • Plasmids / metabolism
  • Protein Binding
  • RNA Folding*
  • RNA, Transfer, Amino Acid-Specific / chemistry*
  • RNA, Transfer, Amino Acid-Specific / genetics
  • RNA, Transfer, Amino Acid-Specific / metabolism
  • Scattering, Small Angle
  • Transfer RNA Aminoacylation*
  • X-Ray Diffraction

Substances

  • Amino Acids
  • RNA, Transfer, Amino Acid-Specific
  • Amino Acyl-tRNA Synthetases