It has been generally accepted that initiation is the rate-limiting step of translation. However, the transition between translational initiation and translational elongation may also play a role in regulating the translational efficiency of a gene, which is defined as the number of proteins produced per mRNA per unit time. In Saccharomyces cerevisiae, genes encoding ribosome subunits, which in general have higher translational efficiency, preferentially use ‘GNN’ as their second codon, suggesting that the mRNA sequence after the start codon may be important to promoter the translational efficiency of a gene. To systematically examine the function of nucleotides after the start codon, we inserted 12 random nucleotides between the start codon and other coding sequences of GFP and measured their impacts on the translational efficiency (GFP level/mRNA level). We observed that GNN containing variants exhibited higher translational efficiency. Further, we performed Ribo-seq experiment to gauge the translational dynamics and found that knocking-out certain initiation factors may eliminate the positive impact of GNN on the translational efficiency, suggesting that GNN promotes translational efficiency by interacting with those initiation factors. Together, our results demonstrate the importance of the transition between translational initiation and translational elongation in regulating translational efficiency and shed light on the genome evolution of the coding sequence context after the start codon.