RNA-protein interactions in assembled editing complexes in trypanosomes.
Multisubunit RNA editing complexes recognize thousands of pre-mRNA sites in the single mitochondrion of trypanosomes. Specific determinants at each editing site must trigger the complexes to catalyze a complete cycle of either uridylate insertion or deletion. While a wealth of information on the protein composition and catalytic activities of these complexes is currently available, the precise mechanisms that govern substrate recognition and editing site specificity remain unknown. This chapter describes basic assays to visualize direct photocrosslinking interactions between purified editing complexes and targeted deletion and insertion sites in model substrates for full-round editing. It also illustrates how variations of these assays can be applied to examine the specificity of the editing enzyme/substrate association, and to dissect structural or biochemical requirements of both the substrates and enzyme complex.
Department of Biochemistry & Biophysics, Texas A&M University, College Station, Texas, USA.
Jorge
J
Cruz-Reyes
D000818:Animals; D001483:Base Sequence; D001671:Biochemistry / Q000379:methods; D002384:Catalysis; D003432:Cross-Linking Reagents / Q000737:chemistry; D017353:Gene Deletion; D008969:Molecular Sequence Data; D011485:Protein Binding; D012313:RNA / Q000737:chemistry*; D017393:RNA Editing; D012322:RNA Precursors / Q000235:genetics; D016053:RNA, Protozoan / Q000235:genetics*; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000378:metabolism*
D003432:Cross-Linking Reagents; D012322:RNA Precursors; D016053:RNA, Protozoan; D012313:RNA