Albany 2001

category image Biomolecular
SUNY at Albany
June 19-23, 2001

An Investigation of Adenine Recognition in the U1A:U1snRNA Complex

RNP (ribonucleoparticle) domain proteins serve a wide range of functions in all eukaryotic cells. These proteins share a "beta1-alpha1-beta2-beta3-alpha2-beta4" secondary structural arrangement. Highly conserved aromatic amino acid residues lie on beta1 and beta3 of these proteins, contacting RNA. In addition to hydrogen bonding, stacking interactions between these aromatic residues and RNA stabilize RNP-RNA complexes. Understanding the contribution of these interactions to the stability of these complexes is key in learning more about the origins of affinity and specificity in RNP-RNA complexes.

The U1A protein:U1 small nuclear RNA (snRNA) complex is a well-characterized model system for the exploration of interactions that stabilize RNP:RNA complexes (Fig. 1). The N-terminal RNP domain of human spliceosomal protein U1A has been shown to bind the stem loop 2 region of the U1snRNA sequence A1-UUGCA6-C with subnanomolar affinity. The two adenines in this sequence have been shown to be biochemically and structurally important in RNP-RNA recognition in the U1A:U1snRNA complex. Crystallographic and NMR studies have shown the presence of a highly specific stacking and hydrogen bonding network between A6 in U1snRNA stem loop 2 and surrounding amino acids in the F56 pocket of the N-terminal RNP of U1A. Moreover, the N1 of A1 hydrogen bonds to highly conserved arginine residues in the U1A Loop 3 region.

Previous studies have shown that substitution of A6 with C, G, and U, and A1 with C significantly destabilizes the complex. We incorporate conservative adenine analogs tubercidin, purine, 1-deazaadenine (c1A), and 4-methylindole into stem loop 2 to determine the effects of disrupting the hydrogen bonding network in the A6/F56 region of the U1A:U1snRNA complex. In addition, c1A was incorporated into the A1 position of the RNA stem loop. We have determined that even small modifications to the hydrogen bonding network in the F56/A6 pocket of the U1A A101:stem loop 2 complex results in 1-2 kcal/mol loss in binding, but there is little effect on stability when c1A is substituted for A1. The results indicate that the hydrogen bonding network in the A6/F56 pocket of the U1A:U1snRNA complex is a major contributor to affinity.

NIGMS, PRF, and NIH support this work.

Jacob B. Tuite, Jerome C. Shiels, Scott J. Nolan, Kerry L. Cecere, and Anne M. Baranger

Department of Chemistry, Wesleyan University, Middletown, CT 06459
Phone: (860) 685-3519; Fax: (860) 685-2211; Email: jtuite@wesleyan.edu