The reduction in TE our website evoked by depletion of eIF4G for small ORF genes is also obvious in the scatterplots of Figure S3, as dampening TE values for the shortest ORF lengths in the eIF4G mutant is observed. Thus, genes with short ORFs tend to be trans lated more efficiently in WT cells and to be dependent on eIF4G for their maximum efficiency. It is noteworthy that the two sets of 100 genes we identified above displaying the greatest changes in TE values on depletion of eIF4G differ dramatically in aver age ORF length. The group exhibiting the greatest reductions in translation efficiency has a mean ORF length below the genome average by nearly a factor of two, while genes showing the greatest increases in efficiency have a mean ORF length 70% larger than average.
These findings suggest that ORF length, in addition to 5UTR length, determines the influence of eIF4G on translational efficiency. Below, we propose a molecular explanation for this finding, based on the known relationship between transcript length and the stability of eIF4F cap interaction. Considering the strong correlation between ORF length and effect of eIF4G depletion on translational efficiency shown in Figure 7, it seems possible that the enrichment of cellular functions associated with the gene sets exhibiting TE4G TEWT 0. 71 or TE4G TEWT 1. 4 described above could at least partially reflect a preponderance of genes with unusually small or large ORF lengths in those functional categories. Discussion In this study, we have examined the genome wide con sequences for translational efficiency of simultaneously eliminating eIF4G2 and depleting eIF4G1 from yeast cells.
The conditional depletion of eIF4G1 achieved using a degron tagged version of this protein was highly effective and reduced the polysome content and rate of translation to only 20 30% of WT levels, indicating a substantial reduction in the rate of translation initiation. We used genome expression microarrays to measure the abundance of each mRNA in heavy polysomes relative to its level in total mRNA to calculate translational efficiencies of 5868 different genes. The results indicated that the over whelming majority of mRNAs experienced only a mod erate change in translational efficiency on eIF4G depletion. Less than 2% of the genes showed a statisti cally significant decrease in TE in the mutant by a factor of 1. 4 of more, and the genes in this group that were affected the most displayed reductions of Batimastat a factor of 2. 5 or less. While the actual percentage of genes affected to this extent is probably higher, only 10% of genes exhibited decreases in TE of this magnitude for each biological replicate, which likely represents the upper size limit for this category.