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Historically, translation termination was viewed simply as the last step inthe process of cellular protein synthesis. Recent findings have changed thisperception dramatically, indicating that events at this stage decide aboutthe fate of the posttermination ribosome and about the degradation of thetranslated mRNA. In particular, it remains to be elucidated how terminatingribosomes are recycled back to start another round of translation. Thecircularisation model of the polysomal mRNA suggests that this ribosomerecycling might be facilitated by 5' – 3' interactions mediated by thecap-binding complex eIF4F and the poly(A) binding protein, Pab1p. Incontrast, downstream of a short upstream open reading frame (uORF) in the 5'untranslated region of a gene, posttermination ribosomes can maintain thecompetence to (re)initiate translation. This study shows that recycling andreinitiation are distinct processes in Saccharomyces cerevisiae. Recyclingvia the 3'-UTR was assessed by restricting ribosome movement along the mRNAusing a poly(G) stretch or the mammalian iron regulatory protein (IRP1)bound to the iron responsive element (IRE). Although 3'-UTR structures werefound to influence translation, the main pathway of ribosome recycling doesnot depend on scanning-like movement through the 3'-UTR. Changes intermination kinetics or disruption of the Pab1 – eIF4F interaction do notaffect recycling, yet the maintenance of normal in vivo mRNP structure isimportant to this process. Using bicistronic ACT1-LUC constructs, elongatingyeast ribosomes were found to maintain the competence to (re)initiate overonly short distances. Thus, as the first ORF to be translated isprogressively truncated, reinitiation downstream of an uORF of 105nucleotides is found to be just detectable, and increases markedly inefficiency as uORF length is reduced to 15 nucleotides. Experiments using astrain mutated in the Cca1 nucleotidyltransferase suggest that the uORFlength-dependence of changes in reinitiation competence is affected bypeptide elongation kinetics, but that ORF length per se may also berelevant. Thus, the loss of the reinitiation potential of elongatingribosomes appears to render these incapable to promote intramolecularrecycling via scanning of the 3'-UTR. |
