Metal ion catalysis during group II intron self-splicing: parallels with the spliceosome.
The identical reaction pathway executed by the spliceosome and self-splicing group II intron ribozymes has prompted the idea that both may be derived from a common molecular ancestor. The minimal sequence and structural similarities between group II introns and the spliceosomal small nuclear RNAs, however, have left this proposal in question. Mechanistic comparisons between group II self-splicing introns and the spliceosome are therefore important in determining whether these two splicing machineries may be related. Here we show that 3'-sulfur substitution at the 5' splice site of a group II intron causes a metal specificity switch during the first step of splicing. In contrast, 3'-sulfur substitution has no significant effect on the metal specificity of the second step of cis-splicing. Isolation of the second step uncovers a metal specificity switch that is masked during the cis-splicing reaction. These results demonstrate that group II intron ribozymes are metalloenzymes that use a catalytic metal ion for leaving group stabilization during both steps of self-splicing. Furthermore, because 3'-sulfur substitution of a spliceosomal intron has precisely the same effects as were observed during cis-splicing of the group II intron, these results provide striking parallels between the catalytic mechanisms employed by these two systems.
Department of Biochemistry and Molecular Biology, Howard Hughes Medical Institute, University of Chicago, Chicago, Illinois 60637, USA.
E J
EJ
Sontheimer
P M
PM
Gordon
J A
JA
Piccirilli
D000494:Allosteric Regulation; D000495:Allosteric Site; D002384:Catalysis; D002413:Cations, Divalent / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D007438:Introns / Q000235:genetics*; D008274:Magnesium / Q000502:physiology; D009690:Nucleic Acid Conformation; D012313:RNA / Q000378:metabolism; D012326:RNA Splicing / Q000502:physiology*; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017381:Spliceosomes / Q000502:physiology*; D013379:Substrate Specificity; D013455:Sulfur / Q000737:chemistry
D002413:Cations, Divalent; D002614:Chelating Agents; D016337:RNA, Catalytic; D012331:RNA, Fungal; C039919:RNA, recombinant; D012313:RNA; D013455:Sulfur; D004492:Edetic Acid; D008274:Magnesium