Reconstitution of hairpin ribozyme activity following separation of functional domains.
The hairpin ribozyme is a 50-nucleotide RNA enzyme of unknown three-dimensional structure. Here, we, demonstrate that interdomain interactions are required for catalytic function by reconstitution of activity following separation of an essential, independently folding domain (loop B) from the substrate binding strand at a helical junction. The resulting construct relies on long range tertiary contacts for catalysis. For this work, we used an optimized ribozyme and substrate, which included sequence changes to minimize the formation of nonproductive conformational isomers. Kinetic analysis was carried out using both single and multiple turnover methods and shows that the catalytic efficiency (kcat/Km) of the reconstituted ribozyme is 10(4)-fold lower than that of the intact ribozyme. The decrease in kcat/Km results entirely from a 10(4)-fold increase in the apparent Km, whereas the kcat parameter is essentially unchanged. Therefore, cleavage chemistry appears to be unimpaired, but the reaction is limited by the productive assembly of the two domains. Our results strongly support a previously proposed model in which the catalytic topology of the ribozyme contains a bend at a helical junction.
Butcher
S E
SE
Markey Center for Molecular Genetics, Department of Microbiology and Molecular Genetics, University of Vermont, Burlington 05405, USA.
Heckman
J E
JE
Burke
J M
JM
D001483:Base Sequence; D001665:Binding Sites; D007202:Indicators and Reagents; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*
D007202:Indicators and Reagents; D016337:RNA, Catalytic