Translation Initiation Factors eIF3 and HCR1 Control Translation Termination and Stop Codon Read-Through in Yeast Cells

摘要

Translation is divided into initiation, elongation, termination and ribosome recycling. Earlier work implicated several eukaryotic initiation factors (eIFs) in ribosomal recycling in vitro . Here, we uncover roles for HCR1 and eIF3 in translation termination in vivo . A substantial proportion of eIF3, HCR1 and eukaryotic release factor 3 (eRF3) but not eIF5 (a well-defined “initiation-specific” binding partner of eIF3) specifically co-sediments with 80S couples isolated from RNase-treated heavy polysomes in an eRF1-dependent manner, indicating the presence of eIF3 and HCR1 on terminating ribosomes. eIF3 and HCR1 also occur in ribosome- and RNA-free complexes with both eRFs and the recycling factor ABCE1/RLI1. Several eIF3 mutations reduce rates of stop codon read-through and genetically interact with mutant eRFs. In contrast, a slow growing deletion of hcr1 increases read-through and accumulates eRF3 in heavy polysomes in a manner suppressible by overexpressed ABCE1/RLI1. Based on these and other findings we propose that upon stop codon recognition, HCR1 promotes eRF3·GDP ejection from the post-termination complexes to allow binding of its interacting partner ABCE1/RLI1. Furthermore, the fact that high dosage of ABCE1/RLI1 fully suppresses the slow growth phenotype of hcr1Δ as well as its termination but not initiation defects implies that the termination function of HCR1 is more critical for optimal proliferation than its function in translation initiation. Based on these and other observations we suggest that the assignment of HCR1 as a bona fide eIF3 subunit should be reconsidered. Together our work characterizes novel roles of eIF3 and HCR1 in stop codon recognition, defining a communication bridge between the initiation and termination/recycling phases of translation. Author Summary Protein synthesis (translation) utilizes genetic information carriers, mRNAs, as templates for the production of proteins of various cellular functions. Typically it is divided into four phases: initiation, elongation, termination and ribosomal recycling. In this article we argue that the strict mechanistic separation of translation into its individual phases should be reconsidered in the light of “multitasking” of initiation factors eIF3, HCR1 and ABCE1/RLI1. In detail, we show that eIF3 and HCR1 not only promote the initiation phase but also specifically act at the other end of the translational cycle during termination. We present genetic and biochemical data linking eIF3 and HCR1 with both eukaryotic release factors (eRF1 and eRF3) and the ribosomal recycling factor ABCE1/RLI1, and propose a model for how all these factors co-operate with each other to ensure stringent selection of the stop codon. Collectively, our findings suggest that changes in one phase of translation are promptly communicated to and coordinated with changes in the other phases to maintain cellular homeostasis of all ongoing processes.