Hong Li, and Song Xue
Department of Chemistry and Biochemistry, Florida State University,
Tallahassee FL
Abstract:
We will describe the efforts in
co-crystallization and molecular replacement solution of an RNA-splicing endonuclease complex. The splicing endonuclease exhibits exquisite
specificity in the recognition of RNA substrates, leading to the alteration
of two RNA phosphodiester bonds. The majority of archaeal splice sites
are located on a pseudosymmetric RNA motif that consists of two three
nucleotide bulges separated by a four-basepair helix (bulge-helix-bulge,
BHB). A number of challenges face the structural studies of the
endonuclease-RNA complex. First, common protein-RNA interaction assays
do not work for this system. Second appropriate chemical modifications
on RNAs are required to inhibit the splicing reaction during crystallization
while maintaining sufficient binding energy of the RNAs for the enzyme.
Thirdly, it is difficult, yet highly desirable, to capture structures of
reaction intermediates by crystallography. We have obtained crystals
of an endonuclease in complex with either an RNA inhibitor or cleavage
products by combining enzyme activity assays and crystallization screening.
Crystals of the current RNA-endonuclease complex diffracted to ~4.0 Å with
severe anisotropy. However, a molecular replacement solution by using
previously determined endonuclease structure could be obtained. Packed
endonucleases in the C2 crystal leave significant packing gaps that are
presumably filled by the bound RNA. Despite the significant progress,
a model for the bound RNA could not be built at the moment because its
electron density appears to be smeared. Close examination on RNA
oligos used for co-crystallization suggests that this is likely due to the
alternating arrangements of the pseudosymmetric BHB RNA with respect to the
crystal lattice. Structure solution of a new crystal containing a 21
mer symmetric BHB RNA and the endonuclease should provide the final proof of
this hypothesis. We hope that our experiences in splicing
endonuclease-RNA complex structural studies will help others who are engaged
in similar studies of RNA-enzyme complexes.
2008 Run
Nov 19th - Dec 22nd
