Translational repression of the Escherichia coli alpha operon mRNA - Importance of an mRNA conformational switch and a ternary entrapment complex

Ribosomal protein S4 represses synthesis of the four ribosomal proteins (in cluding itself) in the Escherichia coli a operon by binding to a nested pse udoknot structure that spans the ribosome binding site. A model for the rep ression mechanism previously proposed two unusual features: (i) the mRNA sw itches between conformations that are "active" or "inactive" in translation , with S4 as an allosteric effector of the inactive form, and (ii) S4 holds the 30 S subunit in an unproductive complex on the mRNA ("entrapment"), in contrast to direct competition between repressor and ribosome binding ("di splacement"). These two key points have been experimentally tested. First, it is found that the mRNA pseudoknot exists in an equilibrium between two c onformers with different electrophoretic mobilities. S4 selectively binds t o one form of the RNA, as predicted for an allosteric effector; binding of ribosomal 30 S subunits is nearly equal in the two forms. Second, we have u sed S4 labeled at a unique cysteine with either of two fluorophores to char acterize its interactions with mRNA and 30 S subunits. Equilibrium experime nts detect the formation of a specific ternary complex of S4, mRNA pseudokn ot, and 30 S subunits. The existence of this ternary complex is unambiguous evidence for translational repression of the alpha operon by an entrapment mechanism.