# The Impact of the Stringent Response on TRAF GTPases and Prokaryotic Ribosome Assembly
## 9 Concluding Remarks  
The innate complexity of 30S and 50S ribosome particle assembly makes it extremely unlikely that stochastic maturation will result in the correct conformation. The propensity for biological systems to enter the most energetically favourable state enhances the need for chaperone proteins responsible for ensuring correct folding of individual proteins, RNAs or, indeed, larger assemblies such as the prokaryotic ribosome. RA-GTPases fulfil this role during ribosome assembly, ensuring that the correct maturation level is achieved before either further r-protein recruitment or subunit joining takes place. There are five major approaches that RA-GTPases use to facilitate ribosome assembly, each of which have been discussed here. 1: Direct non-r-protein regulatory factor recruitment (e.g., YbeY recruitment to the 16S rRNA by Era). 2: Quality control checkpoint before direct r-protein recruitment (e.g., RsgA monitoring the maturity of the decoding centre through interaction with h44; Era preventing S1 docking and 30S PreIC formation prior to 16S processing). 3: rRNA modulation to facilitate further r-protein joining (e.g., RbgA C-terminal domain interacting with 23S rRNA helices to protect and alter their conformation). 4: Disassembly of 70S particles with incorrect rRNA conformations to enable repair and resumption of translation (e.g., Hflx-mediated splitting of heat-damaged 70S ribosomes). 5: Prevention of 70S assembly during unfavourable conditions as a consequence of the stringent response (e.g., Hflx preventing subunit joining when bound to (p)ppGpp; increased mature 50S binding by ObgE during the stringent response). The combination of these results in a high level of control over the modular assembly pathway, enabling coupling of 70S assembly and translational activation to the fluctuating cellular GTP pool at any given time. In addition to the longer-term inhibition of 70S assembly, the stringent response can inhibit the translational efficiency of both initiating and translating 70S ribosomes. This is achieved via targeting of the universally-conserved prokaryotic translation factors bIF-2, EF-Tu, EF-G and RF-3 [109; 110; 111; 112], offering a mechanism of control for each of the major steps of translation: initiation, elongation and termination. All in all, the stringent response exhibits control over every aspect of the ribosomal life cycle, from the inhibition of rRNA synthesis by CodY [141], the inhibition of 16S rRNA processing [13], RA-GTPase-mediated 70S macroassembly [6; 76] and, finally, to an increase in 70S splitting and degradation by Hflx and ObgE [90; 94; 110; 115]. The fact that ribosomes from every known organism require RA-GTPases for correct maturation and, thus, high efficiency in translation highlights the importance of these enzymes. The near-universal conservation of (p)ppGpp amongst prokaryotic organisms presents a novel and interesting target for antibiotic development; it has been long postulated that 70S ribosome assembly is a potentially powerful antimicrobial target [155]. Prevention of 70S assembly and maturation would result in severe reduction in bacterial viability, which may aid in the treatment of potentially multidrug-resistant infections in the years to come. While our understanding of ribosomal biogenesis at the molecular level is improving, it is still unclear what specific function these RA-GTPase enzymes have in ribosomal assembly. Additionally, despite the range of crystallographic and cryo-EM approaches that have been used to study prokaryotic RA-GTPases, the features that distinguish (p)ppGpp-binding RA-GTPases from non-(p)ppGpp-binding RA-GTPases remain elusive. No recognition motif or structural motif has been found to date that can enable the binding of (p)ppGpp, highlighting the need for continued on-going research in this field. Writing--review and editing: [PERSON], [PERSON] and R.M.C. ; supervision: [PERSON].