Evidence that the guanosine substrate of the Tetrahymena ribozyme is bound in the anti conformation and that N7 contributes to binding.
The group I self-splicing introns are RNA enzymes that catalyze phosphodiester-exchange reactions. These ribozymes have a highly specific binding site for guanosine, a substrate for the first self-splicing reaction (Bass & Cech, 1984). The binding site for guanosine has been localized to a specific region of the ribozyme (Michel et al., 1989), but the conformation of the bound guanosine substrate remains unknown. Most analogs of guanosine with substituents at C8 have a preference for the syn conformation; however, some C8-substituted analogs have the potential to form a hydrogen bond between the C8 substituent and the 5'-hydroxyl that would stabilize the anti conformation; we have found that analogs with the potential to form such a hydrogen bond are more active substrates than those that cannot form such a hydrogen bond. These observations led us to test 8-5'-O-cycloguanosine, which is locked in the anti conformation, and 8-(alpha-hydroxyisopropyl)guanosine, which is locked in the syn conformation; the former is active as a substrate, while the latter is inactive. These results strongly suggest that guanosine is bound to the ribozyme in the anti conformation and provide an additional constraint on structural models of this RNA enzyme. We have also examined a series of N7-substituted guanosine analogs; this position had previously been assumed to be unimportant for substrate binding since 7-methylguanosine is an excellent substrate. However, we have found that 7-deazaguanosine and 7-methyl-7-deazaguanosine are less active substrates than guanosine. We discuss several models for the role of N7 in guanosine binding.
Lin
C W
CW
Department of Molecular Biology, Massachusetts General Hospital, Boston 02114.
Hanna
M
M
Szostak
J W
JW
D000818:Animals; D006151:Guanosine / Q000031:analogs & derivatives / Q000737:chemistry*; D066298:In Vitro Techniques; D007700:Kinetics; D009690:Nucleic Acid Conformation; D012326:RNA Splicing; D016337:RNA, Catalytic / Q000737:chemistry* / Q000648:ultrastructure; D013329:Structure-Activity Relationship; D013768:Tetrahymena / Q000201:enzymology*
D016337:RNA, Catalytic; D006151:Guanosine