# The Impact of the Stringent Response on TRAF GTPases and Prokaryotic Ribosome Assembly
## 4 (p)ppGpp-Binding RA-GTPases Involved in 30S Assembly  
### RsgA (YloQ/YjeQ)  
The multidomain GTPase RsgA (YloQ in _B. subtilis_, YjeQ in _E. coli_) is a late-stage 30S ribosome assembly cofactor that is widely conserved among bacteria, although is not essential for growth [6; 51]. It contains the conserved G-motifs arranged in an alternative G4-G5-G1-G2-G3 permutation (Figure 2), making it a member of the circularly-permuted GTPase family (CP-GTPase) alongside the other ribosome biogenesis cofactors YawG, YqeH and RbgA. The switch II region is, thus, located towards the C-terminus of the protein in an unstructured state [52]. Consequently, an additional domain is required for stabilisation that can either be independent or dependent on cofactor binding, and indeed RsgA has an N-terminal OB (oligonucleotide/oligosaccharide-binding) domain and a C-terminal zinc-finger domain, both of which are implicated in binding to RNA (Figure 2) [53]. Despite the availability of the structure of RsgA in multiple nucleotide-bound configurations by both X-ray crystallography and cryo-electron microscopy (cryo-EM) [53; 54; 55], the mechanism of GTP catalysis has not been elucidated due to the instability of the switch I region during crystallography and the low resolution of cryo-EM. It has been proposed that RsgA uses a catalytic residue from the switch I region that is activated upon correct maturation of the h44 of the 16S rRNA [56]. RsgA binds the 30S subunit at the 30S-50S interface with high affinity (\(66.2\pm 7.7\) nM) [57], with the G domain and N-terminal OB domain binding the 30S body and the C-terminal zinc-finger contacting the 30S head (Figure 3) [56; 58]. Binding of RsgA to the immature 30S subunit is associated with the docking of h44 of the 16S rRNA in the correct conformation, leading to the hypothesis that RsgA monitors h44 to ensure correct positioning prior to 30S maturation [53; 54; 56]. Upon binding, the OB  
Figure 2: Domain structure of RA-GTPases inhibited by (p)ppGpp. **(A)** The general domain structure of the highly conserved GTPase domain, featuring the catalytic G1-G5 motifs is shown. Functions for these motifs include: G1 (P-loop, Walker A motif) binds and recognises the \(\alpha\)- and \(\beta\)- phosphate of GTP; G2 and G3 (Switch I and Switch II/Walker B motif, respectively) stabilise the \(\gamma\)-phosphate of GTP through coordination of the Mg\({}^{2+}\) cofactor; G4 confers guanine specificity via \(\pi\)-stacking of the lysine residue with the guanine ring and specific H-bonding of the aspartate with the guanine amino groups; G5 interacts with the guanine via H-bonds, but is poorly conserved and not universally present among TRAF GTPases. Consensus sequences are highlighted. **(B)** The domain architecture of the TRAF GTPase GTPases mentioned in this review. Note the circular permutation of RsgA and RbgA. domain of RsgA is positioned within the A-site of the 30S subunit, with residues F48-H51 inserted into the minor groove of h44. The G domain clamps h44 in close proximity to h24 and h45, stabilising it through interactions with the OB domain and the switch I region of the G domain. The zinc-finger domain binds the 30S head at a position involved in interactions between the charged tRNA and the ribosomal P-site [56]. Taken together, the localisation of RsgA during 30S binding suggests that this protein can monitor the maturation state of the 30S tRNA and mRNA binding sites [56]. Interestingly, these three contact points only occur in the GTP-bound form of RsgA; in the GDP bound form the OB domain is the primary point of interaction. RsgA can also interact with mature 70S ribosomes, resulting in dissociation into the 30S and 50S subunits, while destabilising the r-proteins uS2, uS3, uS7, uS12 and bS21 [56; 59]. The implications of this being that RsgA can rescue kinetically trapped intermediates by encouraging release of r-proteins that have associated incorrectly.