Protein synthesis, performed by ribosomes during mRNA translation, is among the most important processes in a cell. Particularly, correct initiation of translation is crucial to prevent synthesizing incorrect, possibly toxic polypeptides and wasting the cell’s resources. During translation initiation in bacteria, three translation initiation factors (IFs)—IF1, IF2, and IF3—play important roles, including regulating the binding of the two ribosomal subunits, helping the ribosome select the correct initiator tRNA, and more. While researchers have studied IF2 and IF3 extensively and understand each’s function relatively well, the role of IF1 remains nebulous.

This study’s aim was to better understand the function of IF1 and its mechanistic details. To do this, we developed a reliable method to easily generate genomic loss-of-function mutants of IF1, specifically targeting four amino acids in IF1—I44, R45, I46, and R69—because crystal structures show they contact the ribosome during translation initiation, suggesting they may play an important role. We generated ~15 mutants of these amino acids, then measured the resulting effect on the cell’s growth rate. The data, presented here, shows that mutating these four amino acids causes significant growth defects. This suggests the corresponding wild-type amino acids play an important role in IF1’s function, since removing them disrupts its function. Interestingly, several mutants’ growth defects became more severe at lower temperatures, which is the opposite behavior of most protein-folding defects; this implies that the reason for the observed loss-of-function in the mutants is likely more complex, such as a binding defect. Going forward, these results will serve as a guide to investigate the effects of these mutants more closely on a molecular scale, by using single-particle tracking (SPT) to measure the effects of IF1 mutations on the rate of translation initiation. Ultimately, this will help us better grasp the currently poorly understood role of IF1 in translation initiation.