The Role of Initiation Factor 3 Structural Dynamics in Regulating the Fidelity of Translation Initiation

Abstract

Initiation factor (IF) 3 is a protein translation factor that, together with IF1 and IF2, controls the selection of a unique initiator tRNA and the correct messenger RNA start codon by the small (30S) ribosomal subunit and ensures that the subsequent association of the large (50S) ribosomal subunit is selectively accelerated only in response to a correctly initiated 30S subunit. IF3 consists of globular N- and C-terminal domains that are separated by a flexible linker, thus it is a structurally dynamic protein and, perhaps owing to these dynamics, decades of genetic, biochemical, and structural studies have thus far failed to provide a widely accepted mechanism through which IF3 regulates initiator tRNA and start codon selection and 50S subunit association. In order to explore the possible role of IF3 dynamics in controlling these processes, we have constructed an IF3 variant carrying fluorescence resonance energy transfer (FRET) donor and acceptor fluorophores within its globular N- and C-terminal domains and have developed an intramolecular IF3 FRET signal. Using single-molecule FRET imaging, we are using this IF3 signal to investigate the intramolecular dynamics of 30S subunit-bound IF3 during translation initiation. Our results demonstrate that 30S subunit-bound IF3 can access multiple conformational sub-states whose occupancies are regulated by the presence of IF1 and/or IF2 on the 30S subunit as well as by the presence and identity of the tRNA and codon located within the 30S subunit peptidyl-tRNA binding site. Most importantly, we find that a fully assembled and correctly initiated 30S subunit uniquely stabilizes a single IF3 conformational sub-state which we hypothesize serves to permit and/or promote 50S subunit association.