RNA recognition by the joint action of two nucleolin RNA-binding domains: Genetic analysis and structural modeling

Abstract

The interaction of nucleolin with a short stem-loop structure (NRE) requires two contiguous RNA-binding domains (RBD 1 + 2). The structural basis for RNA recognition by these RBDs was studied using a genetic system in Escherichia coli. Within each of the two domains, we identified several mutations that severely impair interaction with the RNA target. Mutations that alter RNA-binding specificity were also isolated, suggesting the identity of specific contacts between RBD 1 + 2 amino acids and nucleotides within the NRE stem-loop. Our data indicate that both RBDs participate in a joint interaction with the NRE and that each domain uses a different surface to contact the RNA. The constraints provided by these genetic data and previous mutational studies have enabled us to propose a three-dimensional model of nucleolin RBD 1 + 2 bound to the NRE stem-loop.