The interdomain linker of Escherichia coli initiation factor IF3: a possible trigger of translation initiation specificity

Initiation factor IF3 is responsible for the accuracy of translation initia tion in bacteria, by destabilizing complexes involving non-initiator tRNA a nd/or nonstart codons. This proofreading is performed on the 30S subunit to which IF3 binds selectively. IF3 has an unusual architecture, with two glo bular domains connected by a mobile, positively charged linker. Here, we ha ve investigated the function of this flexible tether by probing its conform ation when IF3 is bound to the ribosomal RNA. Using site-directed mutagenes is of the linker region, we have also selectively modified its length, its flexibility and its chemical composition. The function of the mutant genes was assayed in vivo, and the structural and biochemical properties of some of the corresponding variant proteins were characterized in vitro. The two isolated domains of IF3 were also cc-expressed in order to test the require ment for their covalent attachment. The results indicate that the physical link between the two domains of IF3 is essential for the function of this p rotein, but that the exact length and chemical composition of the linker ca n be varied to a large extent. A model is presented in which the extended l inker would act as a 'strap', triggering a conformational change in the 30S subunit, which would then ensure initiator tRNA selection.