Selection of the translation start codon is a key step during protein synthesis in human cells. We obtained cryo-EM structures of human 48S initiation complexes and characterized the intermediates of codon recognition by kinetic methods using eIF1A as a reporter. Both approaches capture two distinct ribosome populations formed on an mRNA with a cognate AUG codon in the presence of eIF1, eIF1A, eIF2–GTP–Met-tRNAiMet and eIF3. The ‘open’ 40S subunit conformation differs from the human 48S scanning complex and represents an intermediate preceding the codon recognition step. The ‘closed’ form is similar to reported structures of complexes from yeast and mammals formed upon codon recognition, except for the orientation of eIF1A, which is unique in our structure. Kinetic experiments show how various initiation factors mediate the population distribution of open and closed conformations until 60S subunit docking. Our results provide insights into the timing and structure of human translation initiation intermediates and suggest the differences in the mechanisms of start codon selection between mammals and yeast.

Selection of the translation start codon is a key step during protein synthesis in human cells. We obtained cryo-EM structures of human 48S initiation complexes and characterized the intermediates of codon recognition by kinetic methods using eIF1A as a reporter. Both approaches capture two distinct ribosome populations formed on an mRNA with a cognate AUG codon in the presence of eIF1, eIF1A, eIF2–GTP–Met-tRNAiMet, and eIF3. The ‘open’ 40S subunit conformation differs from the human 48S scanning complex and represents an intermediate preceding the codon recognition step. The ‘closed’ form is similar to reported structures of complexes from yeast and mammals formed upon codon recognition, except for the orientation of eIF1A, which is unique in our structure. Kinetic experiments show how various initiation factors mediate the population distribution of open and closed conformations until 60S subunit docking. Our results provide insights into the timing and structure of human translation initiation intermediates and suggest the differences in the mechanisms of start codon selection between mammals and yeast.

During translation initiation in mammalian cells, 80S ribosomes are assembled at the translation start site on mRNA via a cap-dependent scanning mechanism. The formation of the 80S initiation complex (IC) is essential for proper selection of the start codon and requires concerted action of at least 10 eukaryotic initiation factors (eIFs). This process can be divided into five stages: binding of eIFs to the 40S subunit to assemble the 43S pre-IC (PIC), mRNA recruitment to the 43S PIC, mRNA scanning, 48S IC formation upon start codon recognition, and finally, 80S IC formation after 60S subunit joining. Genetic and thermodynamic studies of translation initiation in lower and higher eukaryotes have revealed the major functions of individual eIFs. However, due to the lack of kinetic analysis, the precise order of events and the regulatory mechanisms underlying IC formation in mammals remain unknown.Here, we show the assembly of human 48S IC in real time using fluorescence-based rapid kinetic techniques in an in vitro reconstituted mammalian translation system. We find that eIF1A binds rapidly to two distinct states of the 40S subunit and stabilizes one of them by a two-population binding-equilibrium model. We also observe that the dissociation of eIF1A from the 40S subunit is slower in the presence of eIF1. By monitoring eIF1A dissociation rates, we are able to systematically analyze the kinetic stability of the partial initiation complexes en route to the 43S PIC or 48S IC. Depending on the combinations of initiation factors bound to the 40S subunit, the complexes take on one or two kinetically distinct states. The 43S PIC and scanning 48S PIC are single-population, thermodynamically and kinetically stable complexes.Upon start codon recognition, the 48S IC resumes its dynamic behavior with two kinetically distinct populations: one dissociating rapidly and the other slowly. Our results uncover key kinetic checkpoints that drive the thermodynamic equilibrium toward complete assembly of the 48S IC by the individual and combined action of initiation factors. This mechanistic understanding of the kinetically favored routes guiding initiation complex formation sets a milestone in deciphering the regulation of protein synthesis in mammalian cells.