The structure of the U-box in the essential Saccharomyces cerevisiae pre-mRNA splicing factor Prp19p has been determined by NMR. The conserved zinc-binding sites supporting the cross-brace arrangement in RING-finger domains are replaced by hydrogen-bonding networks in the U-box. These hydrogen-bonding networks are necessary for the structural stabilization and activity of the U-box. A conservative Val→Ile point mutation in the Prp19p U-box domain leads to pre-mRNA splicing defects in vivo. NMR analysis of this mutant shows that the substitution disrupts structural integrity of the U-box domain. Furthermore, comparison of the Prp19p U-box domain with known RING-E2 complex structures demonstrates that both U-box and RING-fingers contain a conserved interaction surface. Mutagenesis of residues at this interface, while not perturbing the structure of the U-box, abrogates Prp19p function in vivo. These comparative structural and functional analyses imply that the U-box and its associated ubiquitin ligase activity are critical for Prp19p function in vivo.
|Number of pages||6|
|Journal||Nature Structural Biology|
|State||Published - Apr 1 2003|
Bibliographical noteFunding Information:
We thank S. Hatakeyama and K.I. Nakayama from the Department of Molecular and Cellular Biology at Kyushu University for the kind gift of GST-Ubc3. We also gratefully acknowledge J.A. Smith for valuable technical assistance with structure calculations and molecular graphics. Work in our laboratories was supported by the National Institutes of Health in the form of operating grants to K.L.G. and W.J.C., training grant positions to M.D.O., C.W.V.K. and J.A.R., and core facility grants to the Vanderbilt Ingram Cancer Center and the Vanderbilt Center in Molecular Toxicology. K.L.G. is an Associate Investigator of the Howard Hughes Medical Institute.
ASJC Scopus subject areas
- Structural Biology