Biology

Gene

Wobble base pair

Molecular biology

Genetics

Biochemistry

P-site

Chromosomal translocation

Ribosome

Chemistry

Protein biosynthesis

Messenger RNA

Translation (biology)

Transfer RNA

Journal of the American Chemical Society

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 tRNALys, tRNAGlu, and tRNAGln 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 tRNALys affects mRNA decoding and tRNA–mRNA translocation. Using real-time kinetic analysis we show that mcm5-modified tRNALys lacking the s2 group has a lower affinity of binding to the cognate codon and is more efficiently rejected than the fully modified tRNALys. The lack of the s2 modification also slows down the rearrangements in the ribosome–EF-Tu–GDP–Pi–Lys-tRNALys complex following GTP hydrolysis by EF-Tu. Finally, tRNA–mRNA translocation is slower with the s2-deficient tRNALys. These observations explain the observed ribosome pausing at AAA codons during translation and demonstrate ...

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