Transfer RNAs (tRNAs) serve as a dictionary for the ribosome translating the genetic message from mRNA into a polypeptide chain. In addition to this canonical role, tRNAs are involved in other processes such as programmed stop codon readthrough (SC-RT). There, tRNAs with near-cognate anticodons to stop codons must outcompete release factors and incorporate into the ribosomal decoding center to prevent termination and allow translation to continue. However, not all near-cognate tRNAs promote efficient SC-RT. Here, with the help of Saccharomyces cerevisiae and Trypanosoma brucei, we demonstrate that those tRNAs that promote efficient SC-RT establish critical contacts between their anticodon stem (AS) and ribosomal proteins Rps30/eS30 and Rps25/eS25 forming the decoding site. Unexpectedly, the length and well-defined nature of the AS determine the strength of these contacts, which is reflected in organisms with reassigned stop codons. These findings open an unexplored direction in tRNA biology that should facilitate the design of artificial tRNAs with specifically altered decoding abilities.

Department of Biochemistry Gene Center University of Munich Munich Germany

Department of Parasitology Faculty of Science Charles University BIOCEV Vestec Czech Republic

Division of Infectious Diseases Department of Medicine Faculty of Medicine and Dentistry University of Alberta Edmonton Alberta Canada

Faculty of Science Charles University Prague Czech Republic

Faculty of Sciences University of South Bohemia České Budějovice Czech Republic

Institute of Parasitology Biology Centre Czech Academy of Sciences České Budějovice Czech Republic

Laboratory of Regulation of Gene Expression Institute of Microbiology Czech Academy of Sciences Prague Czech Republic

Life Science Research Centre Faculty of Science University of Ostrava Ostrava Czech Republic

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