Abstract: Translation re-initiation provides the molecular basis for translational control of mammalian ATF4 and yeast GCN4 mediated by short upstream ORFs (uORFs) in response to eIF2 phosphorylation. eIF4G is the major adaptor subunit of eIF4F that binds the cap-binding subunit eIF4E and the mRNA helicase eIF4A and is also required for re-initiation in mammals. Here we show that the yeast eIF4G2 mutations altering eIF4E- and eIF4A-binding sites increase re-initiation at GCN4 and impair recognition of the start codons of uORF1 or uORF4 located after uORF1. The increase in re-initiation at GCN4 was partially suppressed by increasing the distance between uORF1 and GCN4, suggesting that the mutations decrease the migration rate of the scanning ribosome in the GCN4 leader. Interestingly, eIF4E overexpression suppressed both the phenotypes caused by the mutation altering eIF4E-binding site. Thus, eIF4F is required for accurate AUG selection and re-initiation also in yeast, and the eIF4G interaction with the mRNA-cap appears to promote eIF4F re-acquisition by the re-initiating 40S subunit. However, eIF4A overexpression suppressed the impaired AUG recognition, but not the increase in re-initiation caused by the mutations altering eIF4A-binding site. These results not only provide evidence that mRNA unwinding by eIF4A stimulates start codon recognition, but also suggest that the eIF4A-binding site on eIF4G made of the HEAT domain stimulates the ribosomal scanning independent of eIF4A. Based on the RNA-binding activities identified within the unstructured segments flanking the eIF4G2 HEAT domain, we discuss the role of the HEAT domain in scanning beyond loading eIF4A onto the pre-initiation complex.