How Initiation Factors Regulate the Subunit Joining Step of Translation Initiation

Abstract

High-fidelity initiator transfer RNA (tRNA) and messenger RNA (mRNA) start codon selection during translation initiation is essential for ensuring the integrity of gene expression; mis-initiation using an elongator tRNA or a sense codon can lead to proteins containing aberrant N-termini or to errors in reading frame selection that yield truncated and/or mis-folded proteins. In bacteria, three essential initiation factors (IFs) bind to the small, 30S, ribosomal subunit and control the accuracy of translation initiation by regulating tRNA and codon selection into the 30S initiation complex (30S IC) and joining of the large, 50S, ribosomal subunit to the 30S IC to form an elongation-competent 70S initiation complex (70S IC). Although the IFs render the rate of 50S subunit joining to the 30S IC dependent on the presence of a correctly selected initiator tRNA that is basepaired to a start codon within the 30S IC, the mechanism through which the IFs accomplish this remains elusive. Using fluorescently labeled IFs, tRNAs, and ribosomal subunits, we have recently developed several single-molecule fluorescence resonance energy transfer signals that report on the conformational dynamics of the 30S IC and on the dynamics of 50S subunit joining to the 30S IC. Here we will present the results of our latest studies, which, when integrated with the available biochemical data, strongly suggest that tRNA- and codon-dependent conformational dynamics of the IFs play a critical role in regulating the 50S subunit joining step of translation initiation.