# The _E. coli_ Global Regulator DksA Reduces Transcription during T4 Infection
## Materials and Methods
### In Vitro Transcription  
In order to check the activity of purified DksA, in vitro transcription assays were performed using a supercoiled template DNA that contains the ribosomal promoter, _PrmBP1_ (_rrnB_ sequence \(-\)180 to +109) [49] as a positive control or a DNA template, _PS17_ (gift of [PERSON]) that contains a \(\sigma^{70}\)-dependent promoter that is not affected by DksA as a negative control. Transcription reactions (10 \(\upmu\)L) were assembled with 5 nM DNA, 30 nM RNAP (final concentration; sigma:core ratio of 2.5:1) that had been reconstituted with or without 250 \(\upmu\)M ppGpp (final concentration), the indicated concentration of DksA, and transcription buffer (50 mM Tris-acetate (pH 8.0), 10 mM magnesium acetate, 10 mM 2-mercaptoethanolhanol, 90 mM potassium glutamate, 10 \(\upmu\)g/mL bovine serum albumin). Solutions were incubated at 37 \({}^{\circ}\)C for 10 min before single round transcription was initiated by the addition of heparin (final concentration of 100 \(\upmu\)g/mL) and ribonucleoside triphosphates (rNTPs; final concentration of 200 \(\upmu\)M each ATP, GTP, CTP and 20 \(\upmu\)M (\(\alpha\)-32P) UTP at 1.7 \(\times\) 10\({}^{4}\) dpm/pmol). After incubation for 10 min at 37 \({}^{\circ}\)C, reactions were collected on dry ice. A solution (10 \(\upmu\)L) containing 10 mM EDTA (pH 7.0), 0.1% bromophenol blue, and 0.1% xylene cyanol in deionized formamide was added. Samples were then heated for 2 min at 95 \({}^{\circ}\)C and 10 \(\upmu\)L aliquots were electrophoresed on 4% polyacrylamide, 7 M urea denaturing gels run in 1\(\times\) TBE (Tris-borate-EDTA). Gels were imaged by autoradiography as described above.  
For in vitro transcriptions with T4 DNA, T4 genomic DNA was purified from concentrated T4 wt by extraction with phenol, phenol:chloroform:isoamyl alcohol (25:25:1), and then chloroform:isoamyl alcohol (24:1); each extraction step was repeated twice. The purified DNA was subsequently dialyzed against TE buffer, and then treated with T4 DNA ligase (New England Biolabs, Ipswich, MA, USA) in the presence of ATP to seal any intramolecular nicks. Transcription reactions (100 \(\upmu\)L) were assembled with 0.06 pmol of T4 DNA, 7.5 pmol pre-reconstituted RNAP (sigma:core ratio of 2.5:1), transcription buffer, and as indicated, 112.5 pmol AsiA, 1.88 pmol MotA and/or 60 pmol DKsA. After incubation at 37 \({}^{\circ}\)C for 10 min, a single round of transcription was initiated by adding heparin (final concentration of 100 \(\upmu\)g/mL) and rNTPs (final concentration of 200 \(\upmu\)M each ATP, GTP, CTP, UTP). Samples were incubated for 10 min at 37 \({}^{\circ}\)C before collection on dry ice. An independent in vitro transcription reaction was performed as described [40] using pGEX-5X-3 DNA (0.05 pmol, Pharmacia Biotech, Piscataway, NJ, USA), which contains the Pt\(\it{ac}\) promoter. Before RNA isolation, a 10 \(\upmu\)L aliquot of the Pt\(\it{ac}\) reaction was added as an internal control into each sample, and then the RNA was extracted and purified by phenol extraction/ethanol precipitation as described above. 5\({}^{\prime}\)-ends of the RNA were detected by primer extension analyses as described above.