Structural perspective on the activation of RNAse P RNA by protein.
Ribonucleoprotein particles are central to numerous cellular pathways, but their study in vitro is often complicated by heterogeneity and aggregation. We describe a new technique to characterize these complexes trapped as homogeneous species in a nondenaturing gel. Using this technique, in conjunction with phosphorothioate footprinting analysis, we identify the protein-binding site and RNA folding states of ribonuclease P (RNase P), an RNA-based enzyme that, in vivo, requires a protein cofactor to catalyze the 5' maturation of precursor transfer RNA (pre-tRNA). Our results show that the protein binds to a patch of conserved RNA structure adjacent to the active site and influences the conformation of the RNA near the tRNA-binding site. The data are consistent with a role of the protein in substrate recognition and support a new model of the holoenzyme that is based on a recently solved crystal structure of RNase P RNA.
Buck
Amy H
AH
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, USA.
Kazantsev
Alexei V
AV
Dalby
Andrew B
AB
Pace
Norman R
NR
D001419:Bacteria / Q000201:enzymology*; D020029:Base Pairing; D001483:Base Sequence; D018983:DNA Footprinting; D024202:Electrophoretic Mobility Shift Assay; D019143:Evolution, Molecular; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D043262:Ribonuclease P / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D012313:RNA; D043262:Ribonuclease P