Asymmetric interactions in the adenosine-binding pockets of the MS2 coat protein dimer

We present here a heterodimer complementation analysis of the contributions of individual A-pocket amino acids to the binding of A-4 and A-10 in different halves of the dimer. Various substitutions of A-pocket residues were introduced into one half of single-chain coat protein heterodimers where they were tested for their abilities to complement Y85H or T91I substitutions (defects in the A-4 and A-10 half-sites, respectively) present in the other dimer half.These experiments provide functional tests of interactions predicted from structural analyses, demonstrating the importance of certain amino acid-nucleotide contacts observed in the crystal structure, and showing that others make little or no contribution to the stability of the complex. In summary, Val29 and Lys61 form important stabilizing interactions with both A-4 and A-10. Meanwhile, Ser47 and Thr59 interact primarily with A-10. The important interactions with Thr45 are restricted to A-4.The interaction of MS2 coat protein with its translational operator is one of the best-understood examples of RNA-protein recognition, having been studied extensively by genetic, biochemical, and structural means [1]. The primary and secondary structures of operator RNA are shown in Figure 1. This RNA hairpin makes contacts with both subunits of a coat protein dimer. One of the most important features of the complex of coat protein with its RNA target is the insertion of two unpaired adenosines into equivalent pockets on different subunits of the coat dimer (Figure 2). The interactions of A-4 and A-10 with coat protein involve non-identical contacts with the same five amino acid residues, namely Val29, Thr45, Ser47, Thr59 and Lys61 [2, 3]. X-ray crystallographic analysis infers specific amino acid-nucleotide contacts, but does not by itself allow a clear definition of their individual contributions to RNA-binding and translational repression. In the experiments described here we introduced amino acid substitutions of A-pocke