An important role of G638 in the cis-cleavage reaction of the Neurospora VS ribozyme revealed by a novel nucleotide analog incorporation method.
We describe a chemical coupling procedure that allows joining of two RNAs, one of which contains a site-specific base analog substitution, in the absence of divalent ions. This method allows incorporation of nucleotide analogs at specific positions even into large, cis-cleaving ribozymes. Using this method we have studied the effects of substitution of G638 in the cleavage site loop of the VS ribozyme with a variety of purine analogs having different functional groups and pK(a) values. Cleavage rate versus pH profiles combined with kinetic solvent isotope experiments indicate an important role for G638 in proton transfer during the rate-limiting step of the cis-cleavage reaction.
Jaikaran
Dominic
D
Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Smith
M Duane
MD
Mehdizadeh
Reza
R
Olive
Joan
J
Collins
Richard A
RA
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009491:Neurospora / Q000201:enzymology* / Q000235:genetics; D009690:Nucleic Acid Conformation; D011685:Purine Nucleotides / Q000737:chemistry; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012331:RNA, Fungal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D011685:Purine Nucleotides; D016337:RNA, Catalytic; D012331:RNA, Fungal; D004722:Endoribonucleases; C438195:varkud satellite ribozyme