A kinetically efficient form of the Chlamydomonas self-splicing ribosomal RNA precursor.
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 previously shown to self-splice. Self-splicing of 23S.3 showed biphasic kinetics, with only approximately 33% reacting efficiently. Removal of a region of the 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 P6a-extension by a 20-nt stem-loop further increased the active fraction to approximately 80%. The k(cat) and K(G)(M) for self-splicing (first step) by these latter RNAs were approximately 1 min(-1) and approximately 20 microM, respectively. These results indicate that Cr.LSU is a highly efficient ribozyme whose folding in vitro is impeded by exonic and/or intronic sequences. The implications for in vivo splicing of Cr.LSU are discussed.
Kuo
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TC
Molecular Cell and Developmental Biology Section, Institute for Cellular and Molecular Biology, Austin, Texas 78712, USA.
Herrin
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D000818:Animals; D001483:Base Sequence; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D017931:DNA Primers; D007700:Kinetics; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing; D012338:RNA, Ribosomal, 23S / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D012322:RNA Precursors; D012338:RNA, Ribosomal, 23S