# The Impact of the Stringent Response on TRAF GTPases and Prokaryotic Ribosome Assembly
## 5 (p)ppGpp-Binding RA-GTPases Involved in 50S Assembly  
### ObgE (CgtA)  
ObgE (CgtA in _B. subtilis_) is an essential GTPase that is highly conserved amongst prokaryotic and eukaryotic organisms. Similar to Era, ObgE is thought to be involved in a plethora of cellular functions including ribosome assembly, modulation of the stress response, sporulation, persistence, cell division, chromosome segregation and DNA replication [63; 89; 90; 91; 92; 93]. The conserved, three domain structure of the Obg family consists of a diverse range of C-terminal domains that are implicated in RNA binding and dimerisation, a central G domain and a highly conserved N-terminal domain that is a structural analogue of tRNA (Figure 2) [93].  
Co-sedimentation assays have revealed that ObgE associates with the 30S subunit weakly and the 50S subunit strongly in the presence of GTP, GMPPNP and the stringent response alarmome ppGpp, with ppGpp increasing the occupancy of the 50S five-fold [93]. Consistently, ObgE has been shown to co-sediment with both the 16S and 23S rRNA in a homodimeric and GTP-dependent manner [94]. Pull-down assays have identified a number of proteins with which ObgE associates, including uS3, uS4, uS5, uS13, bS16, uL2, uL4, uL16, bL17, RNAP \(\beta\)-subunit, the stringent response synthetase/hydrolase SpoT and the RNA helicase CsdA [94]. The N-terminal domain of ObgE is a structural mimic of the tRNA A-site acceptor arm and indeed overlaying the 50S:ObgE and P-site tRNA structures reveals that residues 29-31 of the N-terminal domain of ObgE is capable of contacting the CCA motif of the P-site tRNA [93]. K31 inserts between A76 and S2451 of the tRNA and 23S rRNA, respectively, in a similar way as release factor 2 (RF2), which recognises the termination codon on the template mRNA [93; 95]. Furthermore, the location of ObgE on the 50S (Figure 3) is in close proximity to the methylation sites targeted by RrmJ, RluD and RluC [93], the modification of which is instrumental to the functionality of the 23S rRNA. This methylation is also thought to enhance the GTPase activity of ObgE 120-fold in the presence of the mature 50S subunit [93]. ObgE binding to the 50S also prevented association of the 30S and 30S pre-IC in nucleotide-bound forms. Interestingly, comparison between the IC\({}_{50}\) of ObgE involved in prevention of subunit joining showed that the inhibitory effect is much stronger in the case of the 30S pre-IC than the naked 30S subunit [93], implicating ObgE as a very late-stage assembly cofactor. Taken together, the position of ObgE on the 50S has been interpreted to prevent premature association of tRNAs to the A-site via steric occlusion, as well as monitoring the methylation state of the 23S rRNA prior to GTP hydrolysis and dissociation to enable the initiation of translation. ObgE has also been shown to be capable of 70S splitting in a nucleotide-independent manner when in at least 30-fold excess [93]. However, due to the excess of ObgE used in this experiment, the physiological relevance of this is questionable.  
While ObgE is essential, temperature-sensitive mutants have been produced and used to study the effect of ObgE mutation on rRNA processing in the cell. These mutants form immature dead-end 50S* particles lacking uL16, bL33 and bL34. In ObgE-depleted cells, 16S rRNA processing is severely impaired via prevention of 17S 3' and 5' cleavage and, as a result, the 70S ribosome content of the cell decreases [94]. The interaction between ObgE and CsdA has led to the hypothesis that ObgE recruits CsdA to the immature 16S or 23S rRNA, which is then unwound by the helicase activity of CsdA prior to processing. ObgE is also thought to function as a relatively late stage 50S assembly factor, as it exhibits a lack of association to the 40S intermediates formed in RNA helicase mutant \(\Delta csdA\) and \(\Delta smB\) strains, suggesting it binds after these processing events [96].