Abstract
In bacteria, high A and low G content of the 5' end of the coding sequence (CDS) promotes low RNA stability, facilitating ribosomal initiation and subsequently a high protein to transcript ratio. Additionally, 5' NGG codons are suppressive owing to peptidyl-tRNA drop off. It was, therefore, surprising that the first large-scale transgene experiment to interrogate the 5' effect by codon randomization found the NGG, G-rich codon AGG to be the most associated with high transgene output. Why is this? We show that this is not replicated in other large transgene datasets, where AGG and NGG are associated with low efficiency. More generally, there is limited agreement between the first experiment and others. This we find to be a consequence of non-random construct design. In constructs of the first experiment, AGG disproportionately occurs with non-AGG codons associated with low stability and high protein output, making AGG's association with high output an artefact. While translationally non-optimal codons like AGG are conjectured to slow ribosomes for orderly initiation, we find that in the less biased constructs high, not low, translational adaptation in the first 10 codons is (weakly) predictive of higher translational efficiency. These results have implications for both transgene and experimental design.