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.