Recognition of 30 nucleotide context and stop codon readthrough are determined during mRNA translation elongation.

The nucleotide context surrounding stop codons significantly affects the efficiency of translation termination. In eukaryotes, various 30 contexts that are unfavorable for translation termination have been described; however, the exact molecular mechanism that mediates their effects remains unknown. In this study, we used a reconstituted mammalian translation system to examine the efficiency of stop codons in different contexts, including several previously described weak 30 stop codon contexts. We developed an approach to estimate the level of stop codon readthrough in the absence of eukaryotic release factors (eRFs). In this system, the stop codon is recognized by the suppressor or near-cognate tRNAs. We observed that in the absence of eRFs, readthrough occurs in a 30 nucleotide context-dependent manner, and the main factors determining readthrough efficiency were the type of stop codon and the sequence of the 30 nucleotides. Moreover, the efficiency of translation termination in weak 30 contexts was almost equal to that in the tested standard context. Therefore, the ability of eRFs to recognize stop codons and induce peptide release is not affected by mRNA context. We propose that ribosomes or other participants of the elongation cycle can independently recognize certain contexts and increase the readthrough of stop codons. Thus, the efficiency of translation termination is regulated by the 30 nucleotide context following the stop codon and depends on the concentrations of eRFs and suppressor/near-cognate tRNAs. [ABSTRACT FROM AUTHOR]