Reinitiation and Recycling are Distinct Processes Occurring Downstream of Translation Termination in Yeast

The circularisation model of the polysome suggests that ribosome recycling is facilitated by 5′–3′ interactions mediated by the cap-binding complex eIF4F and the poly(A)-binding protein, Pab1. Alternatively, downstream of a short upstream open reading frame (uORF) in the 5′ untranslated region of a gene, posttermination ribosomes can maintain the competence to (re)initiate translation. Our data show that recycling and reinitiation must be distinct processes in Saccharomyces cerevisiae. The role of the 3′UTR in recycling was assessed by restricting ribosome movement along the mRNA using a poly(G) stretch or the mammalian iron regulatory protein bound to the iron responsive element. We find that although 3′UTR structure can influence translation, the main pathway of ribosome recycling does not depend on scanning-like movement through the 3′UTR. Changes in termination kinetics or disruption of the Pab1–eIF4F interaction do not affect recycling, yet the maintenance of normal in vivo mRNP structure is important to this process. Using bicistronic ACT1-LUC constructs, elongating yeast ribosomes were found to maintain the competence to (re)initiate over only short distances. Thus, as the first ORF to be translated is progressively truncated, reinitiation downstream of an uORF of 105 nt is found to be just detectable, and increases markedly in efficiency as uORF length is reduced to 15 nt. Experiments using a strain mutated in the Cca1 nucleotidyltransferase suggest that the uORF length-dependence of changes in reinitiation competence is affected by peptide elongation kinetics, but that ORF length per se may also be relevant.