A short leader sequence impairs the fidelity of initiation by eukaryotic ribosomes

The functional consequences of unusually short 5' noncoding sequences on eukaryotic mRNAs are explored here by using an in vitro transcription and translation system. As the distance of the first AUG codon from the m7G cap was decreased from 32 to 3 nucleotides, the yield of protein initiated from the first AUG codon progressively decreased, with a corresponding increase in initiation from the second AUG codon. The leakiness attributable to a too-short leader sequence was offset, however, by introducing secondary structure downstream from the first AUG codon.