Flanking sequences with an essential role in hydrolysis of a self-cleavinggroup I-like ribozyme

DIGIR1 is a group I-like ribozyme derived from the mobile twin ribozyme gro up I intron DiSSU1 in the nuclear ribosomal DNA of the myxomycete Didymium iridis. This ribozyme is responsible for intron RNA processing in vitro and in vivo at two internal sites close to the 5'-end of the intron endonuclea se open reading frame and is a unique example of a group I ribozyme with an evolved biological function. DiGIR1 is the smallest functional group I rib ozyme known from nature and has an unusual cope organization including the 6 bp P15 pseudoknot. Here we report results of functional and structural an alyses that identify RNA elements critical for hydrolysis outside the DiGIR 1 ribozyme core moiety. Results from deletion analysis, disruption/compensa tion mutagenesis and RNA structure probing analysis all support the existen ce of two new segments, named P2 and P2.1, involved in the hydrolysis of Di GIR1. Significant decreases in the hydrolysis rate, k(obs), were observed i n disruption mutants involving both segments. These effects were restored b y compensatory base pairing mutants. The possible role of P2 is to tether t he ribozyme core, whereas P2.1 appears to be more directly involved in cata lysis.