Uridine 34 (U-34) at the wobble position of the tRNA anticodon is post-transcriptionally modified, usually to mcm(5)s(2), mcm(5), or mnm(5). The lack of the mcm(5) or s(2) modification at U-34 of tRNA(Lys), tRNA(Glu), and tRNA(Gln) causes ribosome pausing at the respective codons in yeast. The pauses occur during the elongation step, but the mechanism that triggers ribosome pausing is not known. Here, we show how the s(2) modification in yeast tRNA(Lys) affects mRNA decoding and tRNA-mRNA translocation. Using real-time kinetic analysis we show that mcm(5)-modified tRNA(Lys) lacking the s(2) group has a lower affinity of binding to the cognate codon and is more efficiently rejected than the fully modified tRNA(Lys). The lack of the s(2) modification also slows down the rearrangements in the ribosome-EF-Tu-GDP-Pi-Lys-tRNA(Lys) complex following GTP hydrolysis by EF-Tu. Finally, tRNA-mRNA translocation is slower with the s(2)-deficient tRNA(Lys). These observations explain the observed ribosome pausing at AAA codons during translation and demonstrate how the e modification helps to ensure the optimal translation rates that maintain proteome homeostasis of the cell.