Thio-Modification of tRNA at the Wobble Position as Regulator of the Kinetics of Decoding and Translocation on the Ribosome

Abstract

Uridine 34 (U₃₄) at the wobble position of the tRNA anticodon is post-transcriptionally modified, usually to mcm⁵s², mcm⁵, or mnm⁵. The lack of the mcm⁵ or s² modification at U₃₄ 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 s² modification in yeast tRNA^Lys affects mRNA decoding and tRNA-mRNA translocation. Using real-time kinetic analysis we show that mcm⁵-modified tRNA^Lys lacking the s² 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 s² 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 s²-deficient tRNA^Lys. These observations explain the observed ribosome pausing at AAA codons during translation and demonstrate how the s² modification helps to ensure the optimal translation rates that maintain proteome homeostasis of the cell.