Crystallographic analysis of small ribozymes and riboswitches.
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.
Department of Biochemistry and Biophysics, University of Rochester, Rochester, NY, USA.
Geoffrey M
GM
Lippa
Joseph A
JA
Liberman
Jermaine L
JL
Jenkins
Jolanta
J
Krucinska
Mohammad
M
Salim
Joseph E
JE
Wedekind
D002850:Chromatography, Gel; D018360:Crystallography, X-Ray / Q000379:methods*; D004247:DNA / Q000096:biosynthesis / Q000378:metabolism; D004256:DNA Polymerase I / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007474:Ion Exchange; D014176:Protein Biosynthesis; D016337:RNA, Catalytic / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D058928:Riboswitch; D012371:Robotics
D016337:RNA, Catalytic; D058928:Riboswitch; D004247:DNA; D004256:DNA Polymerase I