5' transcript replacement in vitro catalyzed by a group I intron-derived ribozyme.
Group I intron-derived ribozymes can perform a variety of catalytic reactions, including the replacement of the 3' end of a mutant RNA transcript with a corrected version of the transcript [Sullenger, B. A., and Cech, T. R. (1994) Nature 371, 619-622]. We now demonstrate in vitro that a ribozyme, derived from a Pneumocystis carinii group I intron, can replace the 5' end of a targeted exogenous RNA with an endogenous RNA. Our model system is a short synthetic mimic of a k-ras transcript, in which substitution mutations at codon 12 are implicated in a host of cancer types. In these experiments, yields of up to 70% were obtained. We analyzed the length dependence of two molecular contacts, P9.0 and P10, that occur between the ribozyme and the exogenous k-ras mimic, and determined that longer, and thus more stable, interactions result in higher product yields. Furthermore, the length of the loop region L1 can substantially influence the yield and the rate of the reaction. These results are a further demonstration that group I intron-derived ribozymes are quite malleable in terms of intermolecular recognition and catalysis, and that these properties can be exploited in developing potentially useful biochemical tools.
Alexander
Rashada C
RC
Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506, USA.
Baum
Dana A
DA
Testa
Stephen M
SM
D020121:5' Untranslated Regions / Q000235:genetics*; D001483:Base Sequence; D002384:Catalysis; D005091:Exons / Q000235:genetics; D011905:Genes, ras / Q000235:genetics; D006157:Guanosine Monophosphate / Q000235:genetics; D006868:Hydrolysis; D007438:Introns / Q000235:genetics*; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D045363:Pneumocystis carinii / Q000201:enzymology / Q000235:genetics; D012326:RNA Splicing; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics*; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics*
D020121:5' Untranslated Regions; C116167:GIR1 ribozyme; D016337:RNA, Catalytic; D012331:RNA, Fungal; D006157:Guanosine Monophosphate