An unconventional origin of metal-ion rescue and inhibition in the Tetrahymena group I ribozyme reaction.
The presence of catalytic metal ions in RNA active sites has often been inferred from metal-ion rescue of modified substrates and sometimes from inhibitory effects of alternative metal ions. Herein we report that, in the Tetrahymena group I ribozyme reaction, the deleterious effect of a thio substitution at the pro-Sp position of the reactive phosphoryl group is rescued by Mn2+. However, analysis of the reaction of this thio substrate and of substrates with other modifications strongly suggest that this rescue does not stem from a direct Mn2+ interaction with the Sp sulfur. Instead, the apparent rescue arises from a Mn2+ ion interacting with the residue immediately 3' of the cleavage site, A(+1), that stabilizes the tertiary interactions between the oligonucleotide substrate (S) and the active site. This metal site is referred to as site D herein. We also present evidence that a previously observed Ca2+ ion that inhibits the chemical step binds to metal site D. These and other observations suggest that, whereas the interactions of Mn2+ at site D are favorable for the chemical reaction, the Ca2+ at site D exerts its inhibitory effect by disrupting the alignment of the substrates within the active site. These results emphasize the vigilance necessary in the design and interpretation of metal-ion rescue and inhibition experiments. Conversely, in-depth mechanistic analysis of the effects of site-specific substrate modifications can allow the effects of specific metal ion-RNA interactions to be revealed and the properties of individual metal-ion sites to be probed, even within the sea of metal ions bound to RNA.
Shan
S O
SO
Department of Biochemistry, Stanford University, California 94305-5307, USA.
Herschlag
D
D
D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000737:chemistry; D002384:Catalysis; D002413:Cations, Divalent; D004791:Enzyme Inhibitors / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D008969:Molecular Sequence Data; D016337:RNA, Catalytic / Q000037:antagonists & inhibitors* / Q000737:chemistry; D013768:Tetrahymena / Q000201:enzymology*; D013816:Thermodynamics; D013873:Thionucleotides / Q000737:chemistry
D002413:Cations, Divalent; D004791:Enzyme Inhibitors; C116167:GIR1 ribozyme; D016337:RNA, Catalytic; D013873:Thionucleotides; D008345:Manganese; D008274:Magnesium; D002118:Calcium