Details

Autor(en) / Beteiligte

• Thickman, K R
• Swenson, M C
• Kabogo, J M
• Gryczynski, Z
• Kielkopf, CL

Titel

Multiple U2AF super(6) super(5) Binding Sites within SF3b155: Thermodynamic and Spectroscopic Characterization of Protein-Protein Interactions among pre-mRNA Splicing Factors

Ist Teil von

• Journal of molecular biology, 2006-02, Vol.356 (3), p.664-683

Erscheinungsjahr

2006

Link zum Volltext

Quelle

Elsevier ScienceDirect Journals Complete

Beschreibungen/Notizen

• Essential, protein-protein complexes between the large subunit of the U2 small nuclear RNA auxiliary factor (U2AF super(6) super(5)) with the splicing factor 1 (SF1) or the spliceosomal component SF3b155 are exchanged during a critical, ATP-dependent step of pre-mRNA splicing. Both SF1 and the N-terminal domain of SF3b155 interact with a U2AF homology motif (UHM) of U2AF super(6) super(5). SF3b155 contains seven tryptophan-containing sites with sequence similarity to the previously characterized U2AF super(6) super(5)-binding domain of SF1. We show that the SF3b155 domain lacks detectable secondary structure using circular dichroism spectroscopy, and demonstrate that five of the tryptophan-containing SF3b155 sites are recognized by the U2AF super(6) super(5)-UHM using intrinsic tryptophan fluorescence experiments with SF3b155 variants. When compared with SF1, similar spectral shifts and sequence requirements indicate that U2AF super(6) super(5) interactions with each of the SF3b155 sites are similar to the minimal SF1 site. However, thermodynamic comparison of SF1 or SF3b155 proteins with minimal peptides demonstrates that formation the SF1/U2AF super(6) super(5) complex is likely to affect regions of SF1 beyond the previously identified, linear interaction site, in a remarkably distinct manner from the local U2AF super(6) super(5) binding mode of SF3b155. Furthermore, the complex of the SF1/U2AF super(6) super(5) interacting domains is stabilized by 3.3kcalmol super(-) super(1) relative to the complex of the SF3b155/U2AF super(6) super(5) interacting domains, consistent with the need for ATP hydrolysis to drive exchange of these partners during pre-mRNA splicing. We propose that the multiple U2AF super(6) super(5) binding sites within SF3b155 regulate conformational rearrangements during spliceosome assembly. Comparison of the SF3b155 sites defines an (R/K) sub(n)XRW(DE) consensus sequence for predicting U2AF super(6) super(5)-UHM ligands from genomic sequences, where parentheses denote residues that contribute to, but are not required for binding.