# The Impact of the Stringent Response on TRAF GTPases and Prokaryotic Ribosome Assembly
## 5 (p)ppGpp-Binding RA-GTPases Involved in 50S Assembly
### RbgA (YlqF)  
Another late stage 50S assembly cofactor, RbgA (YlqF in _B. subtilis_), is an essential protein in the Firmicutes but is completely absent from Proteobacteria. RbgA displays a two-domain architecture: a circularly permuted N-terminal G domain, which contains an unusual K-loop, and a C-terminal helical ANTAR domain (AmiR and NasR transcription anti-termination regulator) implicated in RNA binding and potential rRNA remodelling (Figure 2) [64; 97]. Interactions between the C-terminal domain and the 23S rRNA leads to protection of nucleotides C928, C942, A2301 and A2354 [98], which are located in helices 38, 38, 81 and 85, respectively. H81 and 85 are positioned in close proximity to the uL5 protein responsible for tRNA interaction within the P-site, so dissociation of RbgA following the stimulation of GTPase activity by the correctly conformed 23S rRNA may enable uL5 docking. Mutation of the ANTAR domain has been shown to negatively impact the GTPase activity of RbgA by preventing 50S interaction. The circular permutation of the G domain is thought to transduce conformational changes to the C-terminal domain, which can influence rRNA binding and, thus, ribosome association in complex with different nucleotides [97].  
Depletion of RbgA in _B. subtilis_ results in the formation of immature large ribosomal subunits which migrate at a density of 45S, however, it is currently unknown whether these intermediates are capable of maturing into the active 50S, or whether depletion of RbgA results in a dead-end non-functional complex [99]. Furthermore, this 45S intermediate is lacking uL16, bL27 and bL36--three r-proteins that contribute to the integrity of the A- and P-site of the 50S subunit, and are postulated to be involved in both A- and P-site tRNA binding [98; 100]. uL16 interacts directly with h38, whereas bL27 interacts with h81, further implicating RbgA in cofactor recruitment following correct 23S maturity. In the presence of GMPNP, RbgA can stably interact with both the 50S and 45S particles (Figure 3), however, when bound to GTP, interactions with the 50S are transient due to 60-fold GTPase stimulation upon binding--indicating a GTP-dependent binding mechanism [99].  
Mutation of amino acid F6 to A in RbgA reduces GTPase stimulation by the 50S subunit 12-fold, and was used to carry out suppressor screening [100]. It was observed that all suppressor mutations mapped to the _rplF_ gene encoding the r-protein uL6, with six unique mutations generated: R3C, G5C, G5S, H66L, T68R and R70P - revealing a groove on L6 which appears to be responsible for suppressing the growth defect of RbgA-F6A. Interestingly, the suppressor strains accumulated novel 44S intermediates, distinct from the 45S intermediates isolated from _rbgA_ mutant strains [100], as well as exhibited an increase in 70S ribosomes as compared to RbgA-F6A. These 44S particles are able to mature in vitro into functional 50S particles, which, despite exhibiting a defect in subunit joining, might explain the suppression of the growth defect present in RbgA-F6A mutants [100]. Despite the absence of direct association between RbgA and uL6, the suppressor mutations are thought to destabilise the uL6-50S interaction, increasing the probability of uL6 association with the correctly oriented 23S rRNA. The role of RbgA may be to properly position the uL6 interaction with h97 and with the sarcin-ricin loop, which then enables tertiary r-proteins uL16, bL27, bL28, bL33, bL35 and bL36 to incorporate. This incorporation could then induce the catalytic step associated with GTP hydrolysis and dissociation of RbgA [100].