# The Impact of the Stringent Response on TRAF GTPases and Prokaryotic Ribosome Assembly
## 6 Interplay between RA-GTPase Activity and the Stringent Response  
Recent genome-wide screening has revealed that (p)ppGpp can bind to, and inhibit the GTPase activity of, the RA-GTPases RsgA, RbgA, Era, HflX and ObgE in both Gram-negative and Gram-positive bacteria (Figure 4) [6; 118], leading to a reduction in the formation of the 70S ribosome and attenuated growth. ppGpp has a higher affinity for these GTPases than GTP, whereas pppGpp and GTP bind with similar strengths [6]. Interestingly, the RsgA homologue in _B. subtilis_ was unable to bind (p)ppGpp [6], potentially due to amino acid differences in the (p)ppGpp binding site. The mechanism of GTPase inhibition is currently uncertain, however, crystal structures of RbgA, the _E. coli_ GTPase BipA and the DNA primase DnaG have been solved in the (p)ppGpp-bound states, revealing that (p)ppGpp acts as a competitive inhibitor via binding to the G domain in a near identical manner to GTP [119; 120; 121; 122]. Binding of ppGpp to the nucleotide-binding pocket of BipA had no effect on the tertiary structure of the enzyme, suggesting that inhibition is purely due to outcompeting GTP binding [120]. Inhibition of DnaG has been studied in much greater detail. The (p)ppGpp binding site overlaps that of the priming RNA NTPs, with the 5\({}^{\prime}\) phosphate tail binding in an identical manner [122]. However, the presence of the 3\({}^{\prime}\) diphosphate in the alarmones enables direct interaction with an associated Mn\({}^{2+}\) cofactor, which consequentially results in a reconfiguration of the guanosine base into an orientation distinct from that of the NTPs. Due to this, it was concluded that inhibition of DNA primase relies on blocking entry of incoming NTPs to the binding site through use of the innate nucleotidyl properties of (p)ppGpp [122]. As a result of the specificity of the binding site of RA-GTPases to guanine rings via stacking of a lysine residue and specific bifurcated hydrogen bonding between an aspartate carboxyl and substrate nitrogen atoms [123], it can be speculated that (p)ppGpp inhibition of these enzymes may take place in a similar manner to that of DnaG. Indeed, the crystal structure of RbgA reveals (p)ppGpp bound in the GTP-binding pocket, however, the position of the switch I (G2) loop differs when compared to the GMPPNP bound form of Lsg1, an RbgA homologue from _Saccharomyces cerevisiae_, with incorrect loop docking taking place in the former. This led to postulation that the \(\delta\) and \(\varepsilon\) phosphate moieties of (p)ppGpp sterically prevent the switch I loop from associating correctly, preventing hydrolysis of (p)ppGpp and prolonging the inhibitory effect [119]. The decrease in GTPase activity of RbgA in the presence of ppGpp [6; 119] may lead to a decrease in RbgA release and, thus, the sequestering of immature 45S and mature 50S ribosomal intermediates. In addition to (p)ppGpp binding, the GTPase Era has been shown to directly associate with the bifunctional (p)ppGpp synthetase/hydrolase Rel\({}_{Sau}\) in _S. aureus_ via the conserved G domain [76]. This interaction increases the GTPase activity of Era, perhaps increasing the dissociation of Era from the 30S during stressed conditions to regulate ribosome assembly. Both Era and Rel\({}_{Sau}\) also interact with CshA, a DEAD-box RNA helicase, potentially as part of an Era-CshA-Rel\({}_{Sau}\) complex. In the presence of Rel\({}_{Sau}\), the helicase activity of CshA was inhibited, thus limiting the impact of this enzyme on ribosomal biogenesis [76]. Despite the likely similarity in (p)ppGpp binding mechanics between HfIX and ObgE and the previously discussed RsgA, RbgA and Era, the resulting effects are very different. Under normal, GTP-bound conditions, HfIX functions as a 100S and 70S splitting factor that can also prevent 30S and 50S subunit joining [115].