The Cryo-EM Structure of a Complete 30S Translation Initiation Complex from Escherichia coli

Abstract

Formation of the 30S initiation complex (30S IC) is an important checkpoint in regulation of gene expression. The selection of mRNA, correct start codon, and the initiator fMet-tRNA ^fMet requires the presence of three initiation factors (IF1, IF2, IF3) of which IF3 and IF1 control the fidelity of the process, while IF2 recruits fMet-tRNA ^fMet. Here we present a cryo-EM reconstruction of the complete 30S IC, containing mRNA, fMet-tRNA ^fMet, IF1, IF2, and IF3. In the 30S IC, IF2 contacts IF1, the 30S subunit shoulder, and the CCA end of fMet-tRNA ^fMet, which occupies a novel P/I position (P/I1). The N-terminal domain of IF3 contacts the tRNA, whereas the C-terminal domain is bound to the platform of the 30S subunit. Binding of initiation factors and fMet-tRNA ^fMet induces a rotation of the head relative to the body of the 30S subunit, which is likely to prevail through 50S subunit joining until GTP hydrolysis and dissociation of IF2 take place. The structure provides insights into the mechanism of mRNA selection during translation initiation.

Author Summary

Translation is the process by which a ribosome converts the sequence of a messenger RNA (mRNA)—produced from a gene—into the sequence of amino acids that comprise a protein. Bacterial ribosomes each have one large and one small subunit: the 50S and 30S subunits. Initiation of translation entails selection of an mRNA, identification of the correct starting point from which to read its code, and engagement of the initial amino acid carrier (tRNA). These events take place in the 30S subunit and require the presence of three initiation factors (IF1, IF2, IF3). Formation of this 30S initiation complex precedes joining with the 50S subunit to assemble the functional ribosome. By using a cryo-electron microscopy approach to visualize the structures without fixation or staining, we have determined the structure of a complete 30S initiation complex and identified the positions and orientations of the tRNA and all three initiation factors. We found that the presence of the initiation factors and tRNA induces rotation of the head relative to the body of the 30S subunit, which may be essential for rapid binding to the 50S subunit and for regulating selection of the mRNA. IF3 had not been seen previously in the context of the 30S structure and its visualization gives insight into a potential role in preventing association of the two ribosomal subunits. These findings are important for understanding how the interplay of elements during the early stages of translation selects the mRNA and regulates formation of functional ribosomes.