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According to common view, AUG recognition by scanning ribosomes sets the final point in start codon selection during eukaryotic translation initiation. On mRNAs containing two or more AUG codons, the establishment of the stable codon-anticodon base paring traps the ribosome at the first AUG in a good context and therefore unambiguously determines the point of translation initiation, while later stages, such as eIF2-bound GTP hydrolysis and 60S subunit joining, have not been reported to select between alternative AUG triplets in mammals. Recently, we showed unexpected distribution of 48S complexes between two closely spaced AUG codons of encephalomyocarditis virus mRNA when translation was blocked by non-hydrolysable GTP analog, GMPPNP. In contrast, arrested 80S complexes reflected the physiological ratio of the two translation products. Here we show that this phenomenon is not limited to the EMCV case, but rather is a common feature of mRNA with two or more nearly located AUG codons. Using the translation initiation reconstitution system combined with kinetic toeprinting, and a set of mRNA constructs we demonstrate that after 48S complex formation at the first AUG codon, under conditions when GTP hydrolysis is impaired, the 40S ribosome is able to resume scanning and slides further to closely spaced downstream initiation codons. In contrast to conventional leaky scanning, such sliding occurs after a rather long pause at the first AUG. Thus, AUG recognition by the anticodon of initiator tRNA per se does not irreversibly lead to the initiation of translation from this particular codon. In contrast, eIF5-induced GTP hydrolysis leading to eIF2-GDP dissociation traps the 48S complex, and such a complex lacking eIF2 is further stabilized by eIF5B and 60S joining. This suggests a novel mechanism of translation regulation under conditions when activity of subunit joining factors is affected Indeed, in cell-free systems we show that changes in eIF5 concentration significantly affect the ratio of polypeptides synthesized from a single mRNA containing two overlapping ORFs, and also regulate translation efficiency of mRNAs with uAUG codon(s). The physiological significance of these observations will be discussed.