Rsp5p is an essential yeast ubiquitin protein ligase that ubiquitinates multiple proteins involved in various processes. Recent studies indicate that ubiquitination also affects translation. Here, we show that the strain with the rsp5-13 mutation exhibits altered sensitivity to antibiotics and a slower rate of translation. Using a sensitive dual-gene reporter system, we demonstrate that stop codon readthrough efficiency is decreased in the rsp5-13 mutant, while both +1 and -1 frameshifting were unaffected. The effect of the rsp5-13 mutation on readthrough could be reversed by increased expression of ubiquitin and partially suppressed by overproduction of the elongation factor eEF1A. As assessed by fluorescence in situ hybridization, the rsp5-13 mutant cells accumulate tRNA nuclear pools, perhaps depleting tRNA from the cytoplasm. Nuclear accumulation of tRNA is observed only when rsp5-13 cells are grown in media with high amino acid content. This defect, also reversed by overproduction of the elongation factor eEF1A, may be the primary reason for altered translational decoding accuracy.