Fluorescence and solution NMR study of the active site of a 160-kDa group II intron ribozyme.
We have reconstructed the group II intron from Pylaiella littoralis (PL) into a hydrolytic ribozyme, comprising domains 1-3 (D123) connected in cis plus domain 5 (D5) supplied in trans that efficiently cleaves spliced exon substrates. Using a novel gel-based fluorescence assay and nuclear magnetic resonance (NMR) spectroscopy, we monitored the direct binding of D5 to D123, characterized the kinetics of the spliced exon hydrolysis reaction (which is mechanistically analogous to the reverse of the second catalytic step of splicing), and identified the binding surface of D123 on D5. This PL ribozyme acts as an RNA endonuclease even at low monovalent (100 mM KCl) and divalent ion concentrations (1-10 mM MgCl(2)). This is in contrast to other group II intron ribozyme systems that require high levels of salt, making NMR analysis problematic. D5 binds tightly to D123 with a K(d) of 650 +/- 250 nM, a K(m) of approximately 300 nM, and a K(cat) of 0.02 min(-1) under single turnover conditions. Within the approximately 160-kDa D123-D5 binary complex, site-specific binding to D123 leads to dramatic chemical shift perturbation of residues localized to the tetraloop and internal bulge within D5, suggesting a structural switch model for D5-assisted splicing. This minimal ribozyme thus recapitulates the essential features of the reverse of the second catalytic step and represents a well-behaved system for ongoing high-resolution structural work to complement folding and catalytic functional studies.
Gumbs
Orlando H
OH
Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.
Padgett
Richard A
RA
Dayie
Kwaku T
KT
D001665:Binding Sites; D024202:Electrophoretic Mobility Shift Assay; D005454:Fluorescence Polarization; D006358:Hot Temperature; D007438:Introns; D007700:Kinetics; D008958:Models, Molecular; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D000459:Phaeophyceae / Q000235:genetics; D012326:RNA Splicing; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012335:RNA, Ribosomal; D012996:Solutions / Q000737:chemistry; D013379:Substrate Specificity
D016337:RNA, Catalytic; D012335:RNA, Ribosomal; D012996:Solutions