Riboswitches pp 115-128 | Cite as

Preparation and Crystallization of Riboswitch–Ligand Complexes

  • Olga Pikovskaya
  • Artem A. Serganov
  • Ann Polonskaia
  • Alexander Serganov
  • Dinshaw J. Patel
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 540)

Summary

Riboswitches are mRNA regions that regulate the expression of genes in response to various cellular metabolites. These RNA sequences, typically situated in the untranslated regions of mRNAs, possess complex structures that dictate highly specific binding to certain ligands, such as nucleobases, coenzymes, amino acids, and sugars, without protein assistance. Depending on the presence of the ligand, metabolite-binding domains of riboswitches can adopt two alternative conformations, which define the conformations of the adjacent sequences involved in the regulation of gene expression. In order to understand in detail the nature of riboswitch–ligand interactions and the molecular basis of riboswitch-based gene expression control, it is necessary to determine the three-dimensional structures of riboswitch–ligand complexes. This chapter outlines the techniques that are employed to prepare riboswitch–ligand complexes for structure determination using X-ray crystallography. The chapter describes the principles of construct design, in vitro transcription, RNA purification, complex formation, and crystallization screening utilized during the successful crystallization of several riboswitches.

Key words:

Purine riboswitch preQ1 riboswitch RNA secondary structure Crystallization NMR spectroscopy 

Notes

Acknowledgment

This research was supported by NIH GM073618.

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Olga Pikovskaya
    • 1
  • Artem A. Serganov
    • 1
  • Ann Polonskaia
    • 1
  • Alexander Serganov
    • 1
  • Dinshaw J. Patel
    • 1
  1. 1.Memorial Sloan-Kettering Cancer CenterDepartment of Structural BiologyUSA

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