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Ribozymes

Volume 848 of the series Methods in Molecular Biology pp 159-184

Date:

Crystallographic Analysis of Small Ribozymes and Riboswitches

  • Geoffrey M. LippaAffiliated withDepartment of Biochemistry and Biophysics, University of Rochester
  • , Joseph A. LibermanAffiliated withDepartment of Biochemistry and Biophysics, University of Rochester
  • , Jermaine L. JenkinsAffiliated withDepartment of Biochemistry and Biophysics, University of Rochester
  • , Jolanta KrucinskaAffiliated withDepartment of Biochemistry and Biophysics, University of Rochester
  • , Mohammad SalimAffiliated withDepartment of Biochemistry and Biophysics, University of Rochester
  • , Joseph E. WedekindAffiliated withDepartment of Biochemistry and Biophysics, University of Rochester Email author 

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Abstract

Ribozymes and riboswitches are RNA motifs that accelerate biological reactions and regulate gene expression in response to metabolite recognition, respectively. These RNA molecules gain functionality via complex folding that cannot be predicted a priori, and thus requires high-resolution three-dimensional structure determination to locate key functional attributes. Herein, we present an overview of the methods used to determine small RNA structures with an emphasis on RNA preparation, crystallization, and structure refinement. We draw upon examples from our own research in the analysis of the leadzyme ribozyme, the hairpin ribozyme, a class I preQ1 riboswitch, and variants of a larger class II preQ1 riboswitch. The methods presented provide a guide for comparable investigations of noncoding RNA molecules including a 48-solution, “first choice” RNA crystal screen compiled from our prior successes with commercially available screens.

Key words

RNA Riboswitches Ribozymes RNA synthesis Purification X-ray crystallography Crystal screen