W0288

Crystal Structural Analysis of an Archaeal RNA Splicing Endonuclease in Three Different Space Groups. Yanming Zhang and Hong Li, Inst. of Molecular Biophysics, Florida State Univ., Tallahassee, FL, 32306.

RNA splicing endonuclease is responsible for excision of introns in transfer RNA and for processing archaeal ribosomal RNAs. The archaeal form of the enzyme recognizes a unique RNA motif that consists of two three-nucleotide bulges separated by a four base-paired helix, or the bulge-helix-bulge motif (BHB motif). A crystal structure of the RNA splicing endonuclease from A. fulgidus (AF) has been reported previously at 2.8 Å (Hong Li & John Abelson: J. Mol. Biol. (2000), 302, 639-648). In the process of co-crystallizing AF endonuclease with a BHB RNA substrate, we have obtained two additional crystals of the AF endonuclease itself. The wild-type enzyme (AFF) was crystallized in space group P2₁ and an N-terminus-truncated form of the enzyme (AFN) was crystallized in space group P2₁2₁2. Both structures were determined by the Molecular Replacement method and refined at 2.5 Å and 2.0 Å respectively. The structure determination process will be described which includes analysis of pseudo-C-centering property in the AFN crystal. Refined AF endonuclease structures from the two new space groups will be compared with that reported earlier in terms of structure differences and the crystal packing environment. The regions of particular interests include the proposed active site, the dimer interface and the proposed RNA-binding surface. Preliminary analysis suggests that the N-terminal domain differs significantly while the C-terminal domain, which includes the proposed active site, differs little among all the structures. Packing analysis of the three crystals forms can suggest new protein engineering strategies for obtaining desired crystal packing.