The 23S rRNA gene of Chlamydomonas reinhardtii contains a group IA3 intron,
Cr.LSU, whose splicing is essential for cell growth. To better understand
Cr.LSU splicing, kinetic analyses were undertaken with 23S.3, a preRNA prev
iously shown to self-splice. Self-splicing of 23S.3 showed biphasic kinetic
s, with only similar to 33% reacting efficiently. Removal of a region of th
e 5' exon that could potentially interfere with the intron core (i.e., P3)
increased the size (53%) of the active fraction. Replacement of the large P
6a-extension by a 20-nt stem-loop further increased the active fraction to
similar to 80%. The k(cat) and K-M(G) for self-splicing (first step) by the
se latter RNAs were similar to 1 min(-1) and similar to 20 mu M, respective
ly, These results indicate that Cr.LSU is a highly efficient ribozyme whose
folding in vitro is impeded by exonic and/or intronic sequences. The impli
cations for in vivo splicing of Cr.LSU are discussed. (C) 2000 Academic Pre
ss.