# The Impact of the Stringent Response on TRAF GTPases and Prokaryotic Ribosome Assembly
## 6 Interplay between RA-GTPase Activity and the Stringent Response
In the ppGpp-bound state, HfIX is able to split the 70S ribosome with comparable activity as the GMPNP-bound state, and can bind to, but remains unable to dissociate from, the 100S hibernating complex due to the lack of GTP hydrolysis, preventing splitting into functional 70S ribosomes [115]. In _S. aureus_, growth at 47 \({}^{\circ}\)C stimulates the activity of HfIX, yet \(\Delta\)_HfIX_ strains are more viable at this higher temperature than the wild type, most likely due to an observed increase in translational efficiency in the knockout. This induction of _liftX_ was alleviated in a \(\Delta\)_sigB_ mutant, with a twofold reduction in expression [115]. A \(\Delta\)_rel\({}_{Sau}\)_ strain of _S. aureus_ derepresses the expression of both HPF and HfIX through an unknown but (p)ppGpp-mediated mechanism [115]. This implicates an accumulation of (p)ppGpp within the cell with increased expression of HfIX, which encourages splitting of the active 70S into inactive 30S and 50S particles. These can then be sequestered by other RA-GTPases in the (p)ppGpp-bound state to arrest translation during low-energy or stressed conditions. Figure 4: The effect of the stringent response on ribosome maturation and translation. During the stringent response the alarmones (p)ppGpp accumulate following synthesis by RSH-superfamily enzymes. In _E. coli_, the interaction between RelA (dark blue) with the ribosome activating complex (RAC) increases the synthesis of (p)ppGpp. The RAC consists of the ribosome (50S, lightblue; 30S, orange), mRNA and uncharged tRNA (green). A charged tRNA is situated in the P site of the ribosome. (p)ppGpp inhibits the GTPase activity of various small GTPases, preventing correct ribosome maturation. (p)ppGpp also inhibits the splitting of inactive 100S ribosome dimers by HfIX but promotes splitting of 70S ribosomes to inactive 50S and 30S subunits. Furthermore, (p)ppGpp inhibits the translational GTPase bIF-2, EF-Tu and RF3, resulting in a reduction in translation. When associated with GTP, ObgE is a 50S maturation factor capable of associating with a range of r-proteins, playing a vital role in PTC development as well as other cellular events, such as DNA replication. During nutrient-rich conditions, the ObgE homologue CgtA from _Vibrio cholera_ can bind to and repress the synthetase activity of the long RSH enzyme SpoT [24]. However, during stringent response activation, ObgE can bind to ppGpp with a similar affinity to GDP as part of a positive feedback mechanism [93]. Co-sedimentation assays showed greater association of ObgE to the 50S when bound to ppGpp rather than GTP or GMPNP [93], corroborating the role of ObgE as an anti-association factor. Shifting the 70S dissociation equilibrium towards the constituent subunits during the stringent response may act as a further mechanism by which translation is stalled and growth is arrested, but may also enable greater subunit degradation, a process which begins via 70S splitting [124]. Interestingly, a growth-suppressed but viable _E. coli obgE::_Tn5 insertion strain had a ppGpp:ppPGpp ratio much lower than seen in the wild type [90], which can be completely reversed by overexpression of native ObgE. ppGpp has been named the major controlling factor that influences growth rate in _E. coli_, suggesting that ObgE has an important role in facilitating the efficacy of the stringent response via conversion of ppPGpp to ppGpp. This process is usually carried out by the GppA 5\({}^{\prime}\)\(\gamma\)-phosphatase, but can also be achieved by the hydrolase SpoT, which has been shown to interact with ObgE in vivo [94; 125]. Studies have shown that a lower ppGpp:ppPGpp ratio has a weaker inhibitory effect on cellular RNA synthesis, potentially due to a reduction in RNAP control by ppGpp [125], which also results in a greater growth rate due to continued transcription of rRNA.