NanGIR1 is a member of a new class of group I ribozymes
whose putative biological function is site-specific hydrolysis
at an internal processing site (IPS). We have previously
shown that NanGIR1 requires 1 M KCl for maximal activity,
which is nevertheless slow (0.03 min–1).
We used in vitro selection and an RNA pool with approximately
nine mutations per molecule to select for faster hydrolysis
at the IPS in 100 mM KCl. After eight rounds of selection,
GIR1 variants were isolated that catalyzed hydrolysis at
300-fold greater rates than NanGIR1 RNA. Although not required
by the selection, many of the resultant RNAs had increased
thermal stability relative to the parent RNA, and had a
more compact structure as evidenced by their faster migration
in native gels. Although a wide spectrum of mutations was
found in generation 8 clones, only two mutations, U149C
and U153C, were common to greater than 95% of the molecules.
These and one other mutation, G32A, are sufficient to increase
activity 50-fold. All three mutations lie within or proximal
to the P15 pseudoknot, a structural signature of GIR1 RNAs
that was previously shown to be important for catalytic
activity. Overall, our findings show that variants of the
Naegleria GIR1 ribozyme with dramatically improved
activity lie very close to the natural GIR1 in sequence
space. Furthermore, the selection for higher activity appeared
to select for increased structural stability.