GROUP I-LIKE RIBOZYMES WITH A NOVEL CORE ORGANIZATION PERFORM OBLIGATE SEQUENTIAL HYDROLYTIC CLEAVAGES AT 2 PROCESSING SITES

A new category of self-splicing group I introns with conserved structu ral organization and function is found among the eukaryotic microorgan isms Didymium and Naegleria. These complex rDNA introns contain two di stinct ribozymes with different functions: a regular group I splicing- ribozyme and a small internal group I-like ribozyme (GIR1), probably i nvolved in protein expression. GIR1 was found to cleave at two interna l sites in an obligate sequential order. Both sites are located 3' of the catalytic core. GIR1-catalyzed transesterification reactions could not be detected. We have compared all available GIR1 sequences and pr opose a common RNA secondary structure resembling that of group I spli cing-ribozymes, but with some important differences. The GIR1s lack mo st peripheral sequence components, as well as a P1 segment, and, at ap proximately 160-190 nt, they are the smallest functional group I riboz ymes known from nature. All GIR1s were found to contain a novel 6-bp p seudoknot (P15) within their catalytic core region. Experimental suppo rt of the proposed structure was obtained from the Didymium GIR1 by RN A structure probing and site-directed mutagenesis, Three-dimensional m odeling indicates a compactly folded ribozyme with the functionally es sential P15 exposed in the cleft between the two principal domains P3- P8 and P4-P6.