SUNY at Albany
June 19-23, 2001
The Determinants of the Specific Recognition of an Adenosine in an RNA-Protein Complex
The U1A protein binds with high affinity and specificity to both stem loop 2 of U1 snRNA and to two internal loops located in the untranslated region (UTR) of its own pre-mRNA. X-ray crystallography of the U1A-stem loop 2 complex as well as the NMR structure of the U1A-UTR complex demonstrated that U1A uses both stacking interactions and a network of hydrogen bonds to recognize an essential adenosine (A6) in the RNA loop region. We have investigated the contribution of each of these interactions towards specific recognition of this adenosine. The phenylalanine that stacks with this adenosine was substituted with aromatic and aliphatic amino acids. In addition, A6 of stem loop 2 was replaced with C, G, and U. If stacking interactions were involved in specificity, then we expected that the mutant peptides would lose their ability to discriminate between wild type and the A6-substituted stem loop 2 target sites. The high affinity and specificity of U1A for its target RNA was maintained in the aromatic mutants. In contrast, the aliphatic mutants bound poorly to RNA and their specificity was diminished from that of the native protein. The results suggest the stacking and hydrogen bonding between RNA bases and the peptide are used in a cooperative fashion in maintaining the specificity of the complex.
Jerome C. Shiels, Jacob B. Tuite, Scott J Nolan, Anne Baranger
Department of Chemistry, Wesleyan University, Middletown CT. 06459