Published online 24 May 2005
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Kinetic analysis of the role of the tyrosine 13, phenylalanine 56 and glutamine 54 network in the U1A/U1 hairpin II interaction
Departments of Biochemistry and Molecular Biology and of Surgery, University of Southern California Los Angeles, CA 90089-9176, USA 1Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California Los Angeles, CA 90089-9176, USA 2Department of Biochemistry and Molecular Biology, Keck School of Medicine, University of Southern California Los Angeles, CA 90089-9176, USA
*To whom correspondence should be addressed. Tel: +1 323 865 0655; Fax: +1 323 865 0158; Email: ilaird{at}usc.edu
Received November 3, 2004. Revised April 15, 2005. Accepted May 1, 2005.
The A protein of the U1 small nuclear ribonucleoprotein particle, interacting with its stem–loop RNA target (U1hpII), is frequently used as a paradigm for RNA binding by recognition motif domains (RRMs). U1A/U1hpII complex formation has been proposed to consist of at least two steps: electrostatically mediated alignment of both molecules followed by locking into place, based on the establishment of close-range interactions. The sequence of events between alignment and locking remains obscure. Here we examine the roles of three critical residues, Tyr13, Phe56 and Gln54, in complex formation and stability using Biacore. Our mutational and kinetic data suggest that Tyr13 plays a more important role than Phe56 in complex formation. Mutational analysis of Gln54, combined with molecular dynamics studies, points to Arg52 as another key residue in association. Based on our data and previous structural and modeling studies, we propose that electrostatic alignment of the molecules is followed by hydrogen bond formation between the RNA and Arg52, and the sequential establishment of interactions with loop bases (including Tyr13). A quadruple stack, sandwiching two bases between Phe56 and Asp92, would occur last and coincide with the rearrangement of a C-terminal helix that partially occludes the RRM surface in the free protein.
Present address: Phinikoula S. Katsamba, Center for Biomolecular Interaction Analysis, School of Medicine, University of Utah, Salt Lake City, UT 84132, USA
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