## 2 Structure of YchF/Ola1 and Its Interaction with Ribosomes  
YchF/Ola1 is a highly conserved three-domain protein (Figure 1), sharing 45% sequence identity and 62% sequence similarity between human Ola1 and _E. coli_ YchF (Figure 2). YchF/Ola1 contains four conserved G-motifs (G1-G4), which defines YchF/Ola1 as a member of the large group of P-loop NTPases [16; 17; 24]. The G1-G3 motifs are located in the N-terminal G-domain, which also contains a conserved serine residue (S16 in _E. coli_) and a widely conserved cysteine residue (C35 in _E. coli_), important for phosphorylation [25] and dimerization [26], respectively (Figure 2). Figure 1: Structural model of _E. coli_ YchF. The structure was retrieved from the Alphafold Protein Structure Databank ([[https://alphafold.ebi.ac.uk/](https://alphafold.ebi.ac.uk/)]([https://alphafold.ebi.ac.uk/](https://alphafold.ebi.ac.uk/)), accessed on 27 November 2021). The ATPase domain is shown in red, the coiled-coil or helical domain in yellow and the TGS-domain in green. Indicated are the four G-motifs (G1–G4), which define and specify nucleotide binding, residue S16, which is phosphorylated, residue N20, which has been mainly used for in vivo site directed crosslinking and residue C35 that is involved in YchF dimerization. The helical domain contains two additional Cys residues in close proximity (C140 & C168), which could act as a redox switch. The G4 motif of YchF/Ola1 in most species differs from the canonical G4 motif in P-loop GTPases and is composed of NxxE instead of NKxD (Figure 2), which is likely responsible for the altered nucleotide specificity of YchF/Ola1 [19]. An exception is YchF of _Haloferax volcanism_, which contains the typical NKxD motif (Figure 2), but the nucleotide specificity of _H. volcanism_ YchF has not been tested so far. The G-domain is followed by a helical domain (coiled-coil domain), which forms together with the C-terminal TGS (ThrRS, GTPase, SpoT)-domain, a large, approx. 20 A wide positively charged cleft, potentially favouring RNA interactions (Figure 1) [19; 27]. The exact roles of the helical and the TGS-domains are unknown. In most proteobacteria, the helical domain of YchF contains two conserved additional cysteine residues (C140, C168 in _E. coli_), which are perfectly positioned for an intramolecular disulphide-bridge [26] (Figures 1 and 2). In _E. coli_, the helical domain was also shown to interact with thioredoxin 1 [26] and thus could act as a redox switch. The TGS-domain is present in many RNA-binding proteins, such as ObgE, Threnoyl tRNA synthetase, (p)ppGpp synthase/hydrolase SpoT and the GTP-pyrophosphinase RelA [28; 12; 29]. TGS-domains belong to the beta-grasp fold superfamily, which also include ubiquitin-like proteins [30], associated with post-translational modifications and proteolysis [31; 32]. Within the TRAF class of GTPases, YchF/Ola1 belongs to the Obg-like protein family, which is associated with ribosome biogenesis and translational control [16; 17; 33]. In _E. coli_ and other enterobacteria, _ychF_ is encoded in one operon together with the gene for the essential peptidyl-tRNA hydrolase Pth [34], which catalyses the removal of peptides from peptidyl-tRNAs that have been prematurely released from the ribosome [35]. This also points to a ribosome-associated role of YchF. Thus, several studies have analysed ribosome binding of YchF/Ola1 and its potential effect on ribosome biogenesis in different organisms [36; 37; 38; 39; 40; 41; 42] (Table 1). A putative role in ribosome biogenesis has only been suggested for _Arabidopsis thaliana_ AtYchF, based on the homology to known ribosome assembly factors, such as ObgE [40], but there is no experimental evidence for an involvement of YchF/Ola1 in ribosome biogenesis so far. Studies in _E. coli_ using a _aychF_ strain or an _ychF_-overexpressing strain did not show any significant effect on the presence and quantities of the 30S, 50S, 70S or polysome fractions [43; 44]. On the other hand, ribosome binding of YchF/Ola1 has been experimentally determined in _E.