In vitro selection of the Naegleria GIR1 ribozyme identifies three base changes that dramatically improve activity

NanGIR1 is a member of a new class of group I ribozymes whose putative biol ogical function is site-specific hydrolysis at an internal processing site (IPS). We have previously shown that NanGIR1 requires 1 M KCI for maximal a ctivity, which is nevertheless slow (0.03 min(-1)). We used in vitro select ion and an RNA pool with approximately nine mutations per molecule to selec t for faster hydrolysis at the IFS in 100 mM KCI. After eight rounds of sel ection, GIR1 variants were isolated that catalyzed hydrolysis at 300-fold g reater rates than NanGIR1 RNA. Although not required by the selection, many of the resultant RNAs had increased thermal stability relative to the pare nt RNA, and had a more compact structure as evidenced by their faster migra tion in native gels. Although a wide spectrum of mutations was found in gen eration 8 clones, only two mutations, U149C and U153C, were common to great er than 95% of the molecules. These and one other mutation, G32A, are suffi cient to increase activity 50-fold. All three mutations lie within or proxi mal to the P15 pseudoknot, a structural signature of GIR1 RNAs that was pre viously shown to be important for catalytic activity. Overall, our findings show that variants of the Naegleria GIR1 ribozyme with dramatically improv ed activity lie very close to the natural GIR1 in sequence space. Furthermo re, the selection for higher activity appeared to select for increased stru ctural stability.