Analysis of the functional role of a G.A sheared base pair by in vitro genetics

A classical genetic strategy has been combined with an in vitro selection method to search for functional interactions between the two domains of the hairpin ribozyme. G(21) is located within internal loop B; it is proposed to form a sheared base pair with A(43) across loop B and to bind a Mg(2+) ion. Both nucleotides are important for ribozyme function, and G.A sheared base pairs are a very widespread motif in structured RNA. We took advantage of its presence in the hairpin ribozyme to study its functional role. Pseudorevertants, in which the loss of G(21) was compensated by mutations at other positions, were isolated by in vitro selection. The vast majority of G(21) revertants contained substitutions within domain A, pointing to functional communication between specific sites within the two domains of the hairpin ribozyme. The possibility of a direct or redundant contacts is supported by electrophoretic mobility shift studies showing that a complex formed between domain B of the ribozyme and the substrate was disrupted and restored by base substitutions that have analogous effects on catalytic activity. The functional significance of this complex, the role of the nucleotides involved, and the basis for magnesium ion requirement is discussed.