Termination and peptide release at the upstream open reading frame are required for downstream translation on synthetic shunt-competent mRNA leaders

We have shown recently that a stable hairpin preceded by a short upstream o pen reading frame (uORF) promotes nonlinear ribosome migration or ribosome shunt on a synthetic mRNA leader (M. Hemmings-Mieszczak and T. Hohn, RNA 5: 1149-1157, 1999). We have now used the model mRNA lender to study further t he mechanism of shunting in vivo and in vitro. We show that a full cycle of translation of the uORF, including initiation, elongation, and termination , is a precondition for the ribosome shunt across the stem structure to ini tiate translation downstream. Specifically, AUG recognition and the proper release of the nascent peptide are necessary and sufficient for shunting. F urthermore, the stop codon contest must not impede downstream reinitiation, Translation of the main ORF was inhibited by replacement of the uORF by co ding sequences repressing reinitiation but stimulated by the presence of th e virus-specific translational transactivator of reinitiation (cauliflower mosaic virus pVI). Our results indicate reinitiation as the mechanism of tr anslation initiation on the synthetic shunt-competent mRNA leader and sugge st that uORF-dependent shunting is more prevalent than previously anticipat ed. Within the above constraints, uORF-dependent shunting is quite tolerant of uORF and stem sequences and operates in systems as diverse as plants an d fungi.