ABSTRACT
In eukaryotes, cap-dependent translation initiation represents one of the most complex processes along the mRNA translation regulation pathway. It results in the formation of several transient complexes involving over a dozen eukaryotic initiation factors (eIFs) and culminates in the accommodation of the start codon at the P-site of the small ribosomal subunit (SSU). In higher eukaryotes, the mRNA sequence in direct vicinity of the start codon, called the Kozak sequence (CRCCaugG, where R is a purine), is known to influence the rate of the initiation process. However, the molecular basis underlying its regulatory role remains poorly understood. Here, we present the cryo-electron microscopy structures of late-stage β-globin 48S IC and histone 4 (H4) 48S IC at overall resolution of 3Å and 3.5Å, respectively. We have prepared our complexes in near-native conditions, directly in cell lysates where initiation factors are present at normal cellular levels, and the composition of our complexes is corroborated by mass spectrometry analysis. Our cryo-EM structures shed light on the fine architecture of the mammalian late-stage (LS) initiation complex (IC) in the presence of “strong” (β-globin) and “weak” (H4) Kozak mRNA consensus sequences. From the high-resolution structures we unravel key interactions from the mRNA to eIF1A, eIF2, eIF3, 18S rRNA, and several ribosomal proteins of the LS48S IC. In addition, we were able to study the structural role of ABCE1 in the formation of native 48S initiation complexes. Our results reveal a comprehensive map of the ribosome/eIFs –mRNA and –tRNA interactions and suggest the impact of mRNA sequence on the structure of the IC after the start-codon recognition. Our high-resolution structures also edify the molecular basis underlying regulation of the Kozak sequence-based translation initiation, including such phenomena as leaky scanning.
