PROJECT SUMMARY/ABSTRACT During protein synthesis, the ribosome integrates multiple cues to ensure that the correct protein is made at the right place, the right time and at the right concentration. These cues are the result of signals triggered by varying cellular needs and environmental conditions such as proliferation and stress. In eukaryotes, the integrated-stress response (ISR) responds to stresses through the activation of kinases that act on the initiation factor eIF2. Phosphorylation of eIF2 represses global translation, but also derepresses translation of key pro-survival mRNAs. In yeast, ISR is activated by the eIF2 kinase Gcn2. Recent studies from several groups, including ours, have pointed to a central role for ribosomes and in particular their stalling during the activation of ISR. Interestingly, ribosome stalling also activates ribosome-quality control (RQC), which depends critically on an E3 ligase Hel2. During the previous funding period, we established that Hel2 is activated in response to ribosome collisions and showed that chemical insults that damage RNA trigger RQC. Notably, these very same agents also activate ISR, suggesting that RQC and ISR are tightly be coordinated. In a very recent study, we showed that not only do ribosome collisions activate both processes, but that the activation of one suppresses that of the other. Emerging from these studies is the observation that collided ribosomes are widely used as sensors to trigger an appropriate response, depending on the type and level of stress. Indeed, in preliminary data presented in this proposal, we provide compelling evidence for a role for ribosome collisions in signaling to other nucleic acid damage pathways, particular those involved in DNA-damage repair. This proposal is focused on understanding the molecular rationale by which collided ribosomes can activate these seemingly unrelated processes. Our preliminary data indicate that the A status of the ribosome is important for ISR activation, and in Aim 1 we will probe the conformation of ribosomes under various stress conditions and assess how they impact Gcn2 recruitment. We will expand on these studies by reconstituting ISR and RQC activities to provide a mechanistic understanding for the apparent preferential activation of RQC over ISR. Notably, robust ISR also requires the presence of the highly conserved transcriptional coactivator Mbf1, which we and others showed to bind collided ribosomes. In Aim 2, we will test the hypothesis that stalling activates ISR using a two-pronged mechanism, in which collided ribosomes in addition to activating Gcn2 modulate ISR coactivation by Mbf1. In particular, we will dissect the role of Mbf1 interactions with the ribosome in regulating its function through post- translational modification. Finally, we have a wealth of preliminary data linking RNA-quality control processes with DNA repair. Aim 3 establishing molecular details about how signaling is transduced between the ...