# The Impact of the Stringent Response on TRAF GTPases and Prokaryotic Ribosome Assembly
## 7 Inhibition of rRNA Transcription and Processing by the Stringent Response  
The stringent response result in a very large change in transcriptional profile in _E. coli_, with over 700 genes differentially expressed, including a decrease in rRNA synthesis and a concurrent increase in expression of amino acid biosynthesis and transport genes [10; 126; 127]. In _E. coli_, (p)ppGpp can bind directly to two sites on the RNAP complex, although ppGpp is the more potent effector (Figure 5) [125; 128]. Site 1 comprises a cleft surrounded by the \(\beta\)' and \(\omega\) subunits of the RNAP, with binding site mutations eliminating the inhibition of transcription in the presence of (p)ppGpp [125; 128; 129]. The ability of an RNAP from a given species to bind to (p)ppGpp at this site can be predicted by the presence of a methionine-alanine-arginine (MAR) motif at the N terminal end of the \(\omega\)-subunit. This MAR motif is conserved amongst Alpha-, Beta-, Gamma- and Deltaproteobacteria but is absent in other classes, such as Firmicutes [16]. The effect of (p)ppGpp on reducing rRNA gene transcription is potentiated by binding of the alarmones to site 2, which is created when the transcription factor DksA binds to the \(\beta\)' subunit of the RNAP [129; 130]. DksA and ppGpp act synergistically, with the effect on RNAP transcription in the presence of both factors much greater than with either one alone. Indeed, both factors are required for the positive activation of amino acid biosynthesis gene promoters during the stringent response [131], as well as the inhibition of transcription from the P1 promoters of all 7 _rrn_ genes in _E. coli_[132]. This is thought to occur via a destabilisation of the short-lived open complexes that form at _rrn_ gene promoters [130; 133]. (p)ppGpp appears also to regulate transcription even in non-stressed conditions. In _E. coli_, low ppGpp levels trigger the activation of the leucine response transcriptional regulator Lrp, whereas high ppGpp levels are required to activate the stress-response RpoS regulon [134]. In Gram-positive bacteria (p)ppGpp does not bind to RNAP and the cofactor DksA is not present. ppGpp has no effect on the stability of the DNA-RNAP open complex in _B. subtilis_ and so cannot affect transcription through the same mechanism as in _E. coli_[135; 136]. Instead, transcription is controlled through GTP levels, which are lower during the stringent response (Figure 5). GTP is used as a substrate in the production of (p)ppGpp, which then acts to directly inhibit GTP biosynthesis. Three enzymes in the GTP biosynthesis pathway are inhibited by (p)ppGpp: the IMP dehydrogenase GuaB; the hypoxanthine phosphoribosyltransferase HarpT; and the guanylate kinase Gmk [136; 137; 138]. This lowered GTP pool contributes to a reduction in transcription by two mechanisms. Firstly, GTP is the initiating nucleotide of _rm_ promoters, so when GTP levels are low in the cell rRNA transcription is downregulated due to a slower initiation rate [135]. This method of rRNA regulation seems specific to Gram-positive bacteria, as the identity of the initiating nucleotide in _E. coli_ has no effect on the promoter regulation by (p)ppGpp [139]. In _S. aureus_, G residues in positions +1-4 can have a role in promoter activity and so any other gene with this property would also be downregulated during the stringent response [20]. In _B. subtilis_, incorrect maturation of either the 3\({}^{\prime}\) or 5\({}^{\prime}\) end of tRNA can induce the stringent response in a manner similar to RelA sensing the uncharged tRNA that accumulates during nutrient starvation [13]. This activation of the stringent response subsequently inhibits 3\({}^{\prime}\) processing of the 16S RNA much more quickly than it does the inhibition of rRNA synthesis, preventing existing ribosomal precursors from maturing into functional 70S complexes [13].