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 preQ(1) riboswitch, and variants of a larger class II preQ(1) 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.

Links

Publisher Full Text

Authors

Lippa GM, Liberman JA, Jenkins JL, Krucinska J, Salim M, Wedekind JE

Institution

Department of Biochemistry and Biophysics, University of Rochester, Rochester, NY, USA.

Source

Methods in molecular biology (Clifton, N.J.) 848: 2012 pg 159-84

MeSH

Chromatography, Gel
Crystallography, X-Ray
DNA
DNA Polymerase I
Electrophoresis, Polyacrylamide Gel
Ion Exchange
Protein Biosynthesis
RNA, Catalytic
Riboswitch
Robotics

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

22315069