## 4 Bacterial Stringent Response
### Stringent Response and the Inactivation of EF-Tu and Translation  
Much of the altered gene expression induced by the stringent response is via changes in transcription, where elevated levels of (p)ppGpp directly bind to RNA polymerase [69; 70] and DNA primase [71] and FtsZ [72]. It has also been determined that the stringent response also alters protein translation by inhibiting translational GTPases including initiation factor IF-2 [73; 74], elongation factors EF-Tu [75] and EF-G [74; 75; 76]. Figure 3 shows a schematic proposed mechanism where aminoacyl-tRNA may evade the translational machinery during the stringent response such that it becomes available for other cellularprocesses such as the L/F transferase mediated targeted proteolysis. During the stringent response, approximately half of the GTP molecules are converted to pentapphosphate guanosine (pppGpp) by RelA and/or SpoT, which are further hydrolyzed by guanosine pentapphosphate phosphatases into tetraphosphate guanosine (ppGpp), the functional molecule of the stringent response [77]. Since EF-Tu requires a GTP molecule to form a ternary complex with aminoacyl-tRNA, the loss of a large number of GTP molecules impairs EF-Tu's ability to bind to aminoacyl-tRNAs efficiently. It has been shown that PPPGpp can substitute GTP and binds to EF-Tu to form the ternary complex with aa-tRNA; however, ppGpp mimics the GDP-bound state of EF-Tu such that EF-Tu:ppGpp cannot form the ternary complex with aminoacyl-tRNAs (\(K_{\mathrm{i}}\) = 7 \(\times\) 10\({}^{-7}\) M) [78]. The presence of the resultant replacement of GDP with ppGpp further leads to an inactive EF-Tu:EF-Ts:ppGpp complex (\(K_{\mathrm{i}}\) = 4 \(\times\) 10\({}^{-5}\) M) [75]. This would effectively trap and inactivate EF-Tu from binding to free aminoacyl-tRNAs, which allows L/F-transferase to bind to free aminoacyl-tRNA for its reaction. Similar tRNA binding abolishing effects by ppGpp have also been observed in initiation factor IF-2 and elongation factor EF-G [73; 74]. Figure 3: A proposed mechanism for tRNA-dependent ribosome-independent peptide bond formation where aminoacyl-tRNA evades from the translational machinery during the stringent response. During nutrient rich conditions, EF-Tu:GDP is rapidly released and exchanged to the GTP-bound state by the guanine nucleotide exchange factor EF-Ts. EF-Tu:GTP then forms a ternary complex with an aminoacyl-tRNA, and delivers the aminoacyl-tRNA to the ribosomal A-site for protein biosynthesis. During nutrient limiting conditions (_i.e._, amino acid starvation), GTP molecules are converted to form (p)ppGpp molecules, which results in less GTP molecules available to form the ternary complex, and, thus, EF-Tu cannot bind to aminoacyl-tRNA effectively. ppGpp molecules have also been shown to inhibit EF-Tu and traps EF-Tu in inactive complexes with EF-Ts or ribosomes. Additionally, potential nutrient-dependent post-translational modifications of EF-Tu may further abolishe tRNA binding, while tRNA\({}^{\mathrm{Leu}}\) (CAG) aminoacylation levels are maintained despite amino acid limited conditions. Together, tRNA\({}^{\mathrm{Leu}}\) (CAG) becomes more available for alternative processes such as the L/F-transferase mediated targeted proteolysis. In addition to the activity of ppGpp, the nutrient status-related post-translational modifications of EF-Tu have also been reported to inhibit its activity. [PERSON] _et al._ showed that, during later phases of growth, the amounts of ribosomal proteins (_i.e._, S1, S2, S7, L7/L12, L9, L19) and elongation factors (Tu, Ts, G) decrease while their phosphorylation levels increase, suggesting phosphorylation may be important in the regulation of translation in several bacterial species [79]. For example, _E.