## 3 Results and Discussion
### RelA Active Site Mutation Studies  
To determine the accuracy of the _in silico_ homology alignments and binding site determinations, two amino acid residues were identified as key to the catalytic activity of RelA, and then tested in the laboratory to ensure their assignment was correct (Figure S1). Tyrosine residues Y-324 and Y-332 (from the alignment) (Figure S1D) were determined to act as one of the largest contributors to the initial binding of GDP or GTP [37]. Y-324 was predicted to stabilize the phosphate of GDP/GTP by  
Figure 2: (p)ppGpp production assay. Qualitative production of (p)ppGpp in PBS buffer under various conditions: Mg\({}^{2+}\) (10 mM and 5 mM), with and without 70S ribosome using \(\gamma\)-\({}^{32}\)P-ATP. hydrogen bonding though means of its hydroxyl group, and Y-332 was predicted to be involved in \(\pi\)-stacking with the guanine's aromatic ring. These stabilizations were predicted to allow for the initial binding of GDP/GTP within the active site. Y-324 and Y-332 residues correspond to the Y-310 and Y-319 residues of the _E. coli_ RelA enzyme. We hypothesized that, if these residues were mutated to alanines (A-310 and A-319), this should bring about a decrease in the catalytic transfer of the pyrophosphate from ATP to form (p)ppGpp. Figure S2 shows the interactions of RelA with the native residues, as well as the lack of interactions when mutated to an alanine residue. To obtain the Y/A-310 and Y/A-319 substitutions of the _E. coli_ RelA, we used two synthetic DNA cassettes to replace the 5' end of the gene in the pJW2755-AM plasmid. The first _PsiI_/_Nsi_I (1144-bp) cassette contained a silent _Xbu_I mutation [43; 44] and the Y/A-310 substitution. The second 365-bp _XbaI_/_Nsi_I cassette introduced the Y/A-319 mutation (Figure S3). The 365-bp region between the _XbaI_ (769) and _Nsi_I (1144) restriction sites contains the predicted RelA active center and can be easily exchanged with a synthetic construct to replace any of the tested amino acids. Two assays were conducted to evaluate the activity of the mutant RelA enzymes--an in vivo (p)ppGpp fluorescent reporter and in vitro (p)ppGpp production assay. The ASKA plasmid _pJW2755-AM_ with the WT RelA protein and its Y/A-310 and Y/A319 versions were transformed into the _E. coli_ AG1 strain containing a pAG001 plasmid, this plasmid contains a _vfp_ gene expressed under a stringent response regulated promoter _PrpsJ_[46]. The _E. coli_ AG1 strain contains a _relA1_ mutation caused by an insertion of an _IS2_ insertion sequence between the 85 th and 86 th codons of the _relA_ gene. These mutants retain a low level of (p)ppGpp synthesis activity [19]. Plasmids pJW2755 AM and its derivatives, as well as pAG001, belong to different incompatibility groups and therefore can co-reside in a single cell. When plasmid encoded RelA expression is induced with isopropyl \(\beta\)-D-1-thiogalactopyranoside (IPTG), the cells produced (p)ppGpp. An increased level of (p)ppGpp decreased the level of YPF synthesis, as this was under the control of the Pr_psJ_ promoter. The results showed a much higher reduction in YFP fluorescence in the case of the WT RelA protein than with its Y/A-310 and Y/A-319 derivatives (Figure 3A). In the in vitro assays, the purified proteins containing the Y/A-310 and Y/A-319, when compared with the WT protein, showed an even more striking reduction in ppGpp production (Figure 3B). These results confirm that the Y-310 and Y-319 amino acid residues play important roles in the enzymatic activity of RelA and, therefore, the active site, as modeled above, can be used as a target for the _in silico_ docking of ligands for the identification of candidate druggable inhibitors. Figure 3: Effect of Y/A319 and Y/A310 substitutions on RelA enzymatic activity. (**A**) Ratio of relative RelA activity of the induced to non-induced cells in an in vivo fluorescence assay. The induction with 1.5 \(\mu\)M IPTG took place at 210 min, indicated with the black triangle. (**B**) In vitro pppGpp production assay.