DISSECTING THE KEY RECOGNITION FEATURES OF THE MS2 BACTERIOPHAGE TRANSLATIONAL REPRESSION COMPLEX

The MS2 RNA operator capsid offers an unparalleled opportunity to stud y sequence-specific protein-protein and RNA-protein interactions in mo lecular detail, RNA molecules encompassing the minimal translational o perator recognition elements can be soaked into crystals of RNA-free c oat protein shells, allowing the RNA to access the interior of the cap sids and make contact with the operator binding sites, Correct interpr etation of these structural studies depends critically on functional a nalysis in solution to confirm that the interactions seen in the cryst al are not an artefact of the unusual approach used to generate the RN A-protein complexes, Here we present a series of in vivo and in vitro functional assays, using coat proteins carrying single amino acid subs titutions at residues which either interact with the operator RNA or a re involved in stabilizing the conformation of the FG loop, the site o f the major quasi-equivalent conformational change. Variant operator R NAs have been assayed for coat protein affinity in vitro. The results reveal the robustness of the operator-coat protein interaction and the requirement for both halves of a protein dimer to contact RNA in orde r to achieve tight binding, They also suggest that there may be a dire ct link between the conformation of the FG loop and RNA binding.