# The _Ins_ Gene of _Escherichia coli_ Is Transcriptionally Down-Regulated by (p)ppGpp
## 3 Results
### High Levels of (p)ppGpp Directly Repress lns Promoter  
According to our previous observations [30], we tested the effect of high amounts of (p)ppGpp, linked to amino acid starvation, on the promoter activity of _lns_. To this aim, the intracellular (p)ppGpp levels were artificially increased by overexpressing the RelA alpha fragment in plasmid pKT31, carrying the (p)ppGpp catalytic domain independent of ribosome binding. The isogenic plasmid pKT40, because of the insertion of a stop codon in _relA_ gene, encoded a truncated protein and was used as control. The production of functional RelA and its inactivated variant from pKT plasmids was achieved by adding IPTG to the cell cultures [35]. The nutritional upshift, as consequence of moving stationary-phase cells to a fresh medium not supplemented with IPTG, causes an abrupt burst (\(\sim\)threefold) of _lns_ transcription (Figure 3A). Conversely, the accumulation of (p)ppGpp from pKT31 in induced cells, as determined by thin layer chromatography (Figure 3B,D), completely abolishes the transcriptional activity of _lns_ promoter (Figure 3A). As control, the expression of a nonfunctional RelA enzyme from pKT40 does not affect the _lns_ mRNA level regardless of IPTG induction (Figure 3C). These results suggest that (p)ppGpp exerts a targeted repressive action on _lns_ promoter, encouraging us to better investigate the effect of (p)ppGpp on _lns_ transcription through an in vitro purified system as a function of increasing concentrations of this alarmone. As shown in Figure 4A,B, the addition of 0.5 mM of (p)ppGpp results in a residual _lns_ transcription of \(\sim\)10% and _lns:cat_ mRNA becomes undetectable when this regulatory nucleotide is present at 1 mM, value that corresponds to the concentration estimated in vivo after amino acids starvation [42]. Importantly, suboptimal concentrations of (p)ppGpp (below 0.5 mM) that are not able to completely shut down the hns promoter activity, indeed causes the appearance of truncated cat transcripts (Figure 4A) as products of premature arrests of mRNA synthesis. This is possibly due to the pausing of RNA polymerase at transcriptionally unfavorable regions. [PERSON] et al. [9; 10] demonstrated that (p)ppGpp was able to regulate transcription initiation from sensitive promoters by directly interacting with RNA polymerase. Thus, according to those findings and our in vitro transcription results (Figure 4), we explored the effects of (p)ppGpp on the interaction of RNAP with the promoter region of _lns_. The yield of DNA-RNAP complexes was investigated by electrophoretic mobility shift assay (EMSA) performed as a function of increasing periods of time without and with a fixed concentration of (p)ppGpp (Figure 5A). To convert unstable complexes into stable initiation complexes, the start ribonucleotide ATP was added. The RNAP-_lns_ promoter binding kinetic was followed in a time course experiment and stopped using heparin, which, by irreversibly interacting with the free enzyme, prevented the aggregation of additional DNA-RNAP complexes [43]. As shown in Figure 5B, (p)ppGpp considerably slows down the formation of RNAP-_lns_ promoter complex that is reflected in at least threefold reduction in the association constant (K\({}_{\text{a}}\)). Summarizing, EMSA results suggest that (p)ppGpp negatively affects the extent of complexes trapped in transcription that are therefore considered to be productive, hence accounting for transcriptional inhibition of _lns_ promoter by (p)ppGpp as observed both in vivo and in vitro as well (Figures 3 and 4). Figure 3: High levels of (p)ppGpp inhibit _lns_ transcription in vivo. The level of _lns_ transcript was monitored during growth in Phosphates-free medium at 37 \({}^{\circ}\)C in _E. coli_ cells (KT793) expressing (**A**) or not (**C**) an active ReLA synthetase.