# The _E. coli_ Global Regulator DksA Reduces Transcription during T4 Infection
## 4 Discussion  
DksA, a transcriptional regulator that modulates gene expression in _E. coli_ at the levels of transcription initiation [(63)] and elongation [(15; 64)], belongs to a family of proteins that can insert themselves into the 2\({}^{\circ}\) channel of RNAP, the channel from which rNTPs enter the active site. The best characterized system of DksA regulation is stress response: the repression of ribosomal promoters  
Figure 5: Presence of DksA has no effect on transcription from various T4 Pe’s in vitro. Representative gels show primer extension products for Pe40.4 (**a**), Pm_46_ (**b**), and Pm_dsbA_ (**c**) arising from in vitro transcription of T4 wt DNA in the presence of DksA, MotA, and/or AsiA, as indicated. Each primer extension reaction also contained Pt_ac_ RNA and its primer as an internal control. In vivo RNA isolated from a T4 wt infection of B606 wt at 5 min post-infection was used as a positive control for each primer (labeled as lane C). The lengths of primer extension products are indicated in nt. and the activation of promoters for amino acid biosynthesis genes during amino acid starvation [65]. Both inhibition and activation typically involve the small molecule ppGpp [21], whose level rises dramatically during stress response, even as the level of DksA remains relatively constant [13; 14]. For instance, the presence of ppGpp significantly improves the ability of DksA to inhibit initiation from ribosomal promoters [13]. DksA is a key player in bacterial survival under various environmental changes. For instance, _\(\Delta\)dksA Salmonella enterica_ is hyper-susceptible to the bacteriostatic effects of nitric oxide free radicals and is attenuated in macrophage and murine models of infection [66; 67]; _\(\Delta\)dksA Shigella flexneri_ has decreased Hfq transcription, causing the loss of virulence [68]; and in _E. coli_, DksA has been shown to be important for survival of dehydration [69]. More recently, various studies have shown that DksA provokes global changes in transcriptional expression in host cells under various stresses, such as nitrosative, oxidative and nutrient stresses [70; 71]. To our knowledge, no study has reported whether DksA contributes defense systems by modulating gene expression of phages during infection. Bacteriophage T4 is a good model system for investigating this since it primarily regulates gene expression at the level of transcription. Temporal gene expression by T4 is controlled by the recognition of three distinct promoter architectures (early, middle, and late). Pe's contain very strong \(\sigma^{70}\) consensus sequences (\(-10\) and \(-35\) elements) that are immediately recognized by the host transcriptional machinery [23; 72]. Pm's and Pl's require viral-encoded factors to modify the sequence specificity of host RNAP. Middle genes are expressed approximately 1 to 2 min post-infection at 37 \({}^{\circ}\)C by the activation of Pm's and/or extension of transcripts from Pe's (reviewed in [23]). Activation of Pm's requires two T4 early proteins, MotA and AsiA, which together modify the specificity of host RNAP/\(\sigma^{70}\) holoenzyme, allowing it to recognize a specific \(-30\) sequence (the MotA box) rather than the host \(-35\) element. Late transcription is initiated approximately 5-7 min after infection by the activation of Pl's. These promoters do not contain either the bacterial \(-35\) element or the MotA box sequence, but instead contain a novel \(-10\) sequence, known as the late TATA box, that requires a new sigma factor encoded by T4 gene \(55\), as well as the activator encoded by gene 33 [24]. In addition, late transcription requires active T4 DNA replication through the activity of the T4 protein gp45, which is the replication sliding clamp that also serves as an enhancer of late transcription [24]. The major finding of this study is that host protein DksA down-regulates the activity of Pe's, thereby limiting the number of progeny produced during a single infection. Consequently, DksA serves to suppress T4 infection.