# The Impact of the Stringent Response on TRAF GTPases and Prokaryotic Ribosome Assembly
## 5 (p)ppGpp-Binding RA-GTPases Involved in 50S Assembly
### HflX  
Previously characterised as both an ATPase and GTPase, the universally conserved HflX has been implicated in manganese homeostasis, ribosome binding, 70S splitting and even RNA secondary structure unwinding [65; 101; 102; 103; 104; 105]. Two isoforms of HfIX exist in nature, the first is found in organisms such as _E. coli_ and _S. aureus_ and consists of a three domain architecture--the conserved N-terminal HfIX domain, the central G domain and the C-terminal domain of unknown function (Figure 2) [104]. The second, atypical isoform lacks the C-terminal domain and has been found in the hyperthermophilic archaeon _Sulfolobus solfataricus_[106]. A helical linker region connects the N-terminal domain and G domain. Interestingly, the HfIX N-terminal domain contains a unique nucleotide binding fold, capable of binding ATP without the use of a canonical Walker A nucleotide binding motif [107]. Several years later this domain was shown to exhibit ATP-dependent RNA helicase activity, for which the helical linker is crucial [104]. This linker domain interacts with domain V of the 23S rRNA following a dramatic, 20 A conformational change in response to ATP binding, which is a prerequisite for rRNA unwinding [104]. The ATPase activity is stimulated by an aberrant form of the 50S, generated in vitro by heat shock and 1.5 M NaCl washing. As such, it is logical to assume that one role of HfIX is to repair incorrectly folded rRNA during the assembly process [104]. It has also been demonstrated that HfIX is a heat-shock protein encoded in an operon downstream of the gene encoding the universal stress response protein Hfq, which is under the control of a heat sensitive promoter in _E. coli_[108; 109; 110]. Cells lacking Hfq contain higher levels of immature 30S ribosomes compared to the wild type, suggesting the general stress response is linked to 30S assembly via this protein [111; 112]. This may imply that HfIX could have a role in the repair of abnormal 50S subunits generated during conditions of heat stress. Contradictory reports have shown that HfIX can bind to the 30S, 50S and 70S ribosomal particles in the presence or absence of guanosine nucleotides, with ribosome binding enhancing the stability of the nucleotide-bound state [103; 106; 113]. To clarify the issue, [PERSON] et al. carried out isopycnic ultracentrifugation of HfIX with each subunit in the presence of different nucleotides [65], revealing an enrichment of 50S association when bound to GMPPNP, with dissociation requiring GTP hydrolysis. A similar nucleotide-dependence was observed for 70S binding, accompanied by a strong splitting activity of both the isolated 70S particle and puromycin-treated polysomes, with GMPPNP-bound HfIX splitting 10-fold more efficiently than the apo GTPase [65]. Notably, the presence of peptidyl-tRNA in the ribosomal P-site, close to the HfIX binding site, partially inhibited GTPase activity [110; 65]. This suggests the preferred substrate of HfIX splitting is translationally stalled 70S, containing a de-acylated tRNA in the P-site that is characteristic of translationally-stalled ribosomes. Subunit recycling by HfIX could be involved in the rescue of these stalled ribosomes. Cryo-EM structures reveal that the binding site of HfIX is in fact the subunit interface (Figure 3), with the N-terminal helicase domain protruding towards the PTC and contacting several rRNA helices. This provides a hypothesis as to why GTP hydrolysis is not required for 70S splitting, as HfIX binding disrupts bridge B2a between h69 and the 16S rRNA towards the 3\({}^{\prime}\) of h44 [114] in a similar manner to RRF (Ribosome Recycling Factor) during standard post-translational recycling [65]. It is, therefore, apparent that HfIX can act as both an anti-association factor, inhibiting 70S formation until GTP hydrolysis removes HfIX from the 50S, and as a splitting factor, rescuing stalled ribosomes under stress [65].