## 4 The Role of YchF/Ola1 in Translation  
The available data support the primary role of YchF/Ola1 in regulating the proteostasis network when cells encounter stress conditions [81; 82]. This includes translational regulation at the level of translation initiation [39; 41; 58; 42] and regulation of protein degradation [59; 73; 69]. Observations such as decreased cell migration, increased cell matrix adhesion [57; 62] and reduced cell cycle progression [58] in the absence of Ola1 are likely either directly or indirectly linked to its role in proteostasis regulation. Translation is primarily controlled at the energy-consuming initiation step, which is significantly different between eukaryotes and bacteria [83; 84; 85]. In eukaryotes, initiation starts with the formation of a ternary complex, consisting of eIF2-GTP, and the aminoacylated tRNAMet (elF2-GTP-Met-tRNAMet; Figure 4A). In conjunction with the 40S ribosomal subunit and additional initiation factors (elF1, eIF2A, eIF3 and eIF5) the ternary complex forms a 43S pre-initiation complex, which binds the activated mRNA. mRNA activation involves specific recognition of the m7G-Cap structure at the 5'end of the mRNA by the eIF4F complex. The resulting 48S complex then scans the mRNA for identifying the start codon. Once the Met-tRNAMet has recognized the AUG start codon, eIF2 and other initiation factors are released, enabling 60S subunit binding, resulting in the elongation competent 80S ribosome [86; 87]. This Cap-dependent initiation is primarily regulated at the level of ternary complex formation via inactivation of eIF2 [88]. Figure 4: Model of translational regulation by Ola1/YchF. (**A**) In eukaryotes, Ola1 binds to eIF2 and stabilizes eIF2 in its GDP-bound state, which would subsequently reduce the formation of the ternary complex. As a consequence, alternative translation initiation mechanisms are favoured, leading to selective synthesis of stress-response proteins. (**B**) In prokaryotes, such as _E. coli_, YchF binds preferentially to the 30S ribosomal subunit and regulates IF3-binding to the 30S subunit, which favours canonical translation initiation. Upon stress conditions, _ychlF_ expression is reduced and the ATPase activity of YchF is further inhibited by dimerization. The absence of active YchF possibly favours IF3 binding to the 50S subunit and stimulates leaderless mRNA translation. Note that YchF was also found in contact with the 50S ribosomal subunit; this interaction is likely only transient and not depicted here, although it might be involved in regulating IF3 binding to the ribosome. TrxA corresponds to thioredoxin A, which was shown to dissociate the inactive YchF dimer into active YchF monomers [26]. See text for further details. Ola1 was shown to prevent ternary complex formation via binding to eIF2 [39], which would subsequently reduce protein synthesis (Figure 4A). Reduced formation of the ternary complex is a well-studied consequence of the activated integrated stress response (ISR) [89]. Activation of multiple kinases by the ISR leads to stress-induced inhibition of eIF2 via phosphorylation [86]. Ola1 is suggested to provide an alternative mechanism for reducing ternary complex formation by stabilizing the eIF2-GDP complex (Figure 4A), which would prevent further initiation events [39; 86]. Although Cap-dependent translation is common under non-stress conditions, eukaryotic cells also engage Cap-independent translation mechanisms when they are exposed to stress [86; 90; 91]. One example for a Cap-independent translation mechanism is the translation of the cAMP-dependent transcription factor ATF4, which is produced by a translation re-initiation mechanism in response to eIF2 phosphorylation [92; 93; 90]. Stimulated ATF4 production as a consequence of reduced ternary complex formation in the presence of Ola1 would then promote transcription of genes linked to oxidative stress, ER stress or nutrient limitation [94; 95; 96].