E coli rna polymerase core enzyme

In vitro transcription assays were performed using the E. coli RNA Polymerase Core enzyme (Epicentre, Madison, WI) and the purified rPG0162 protein. The DNA fragment containing the promoter region of the PG0162 gene was used as a template. The reaction mix which contained 40 mM Tris-HCl (pH 7.5), 150 mM KCl, 10 mM MgCl₂, 0.1 mM DTT, 0.01% Triton X-100, 2.5 mM each of NTP mix, ∼1 mCi [α-32P]-ATP (MP Biomedicals, Solon, OH) was incubated at 37 °C for 2 hr. Samples were analyzed on a denaturing urea polyacrylamide gel and quantified using the phosphor imaging system (Molecular Dynamics, Sunnyvale, CA).

In vitro transcription assay was performed using the E. coli RNA polymerase core enzyme (Epicentre, Madison, WI), and the purified rPG1660 protein. The DNA fragment carrying the PG1660 promoter was used as template. The primers used are listed in Table 2. The reactions were incubated at 37°C for 2 h in buffer containing 40 mM Tris–HCl (pH 7.5), 150 mM KCl, 10 mM MgCl₂, 0.1 mM dithiothreitol, 0.01% Triton X-100, 2.5 mM of NTP mix, ~1 mCi [α-³²P]-ATP (MP Biomedicals, Solon, OH). The samples were analyzed on an 8% polyacrylamide denaturing gel and quantified using a phosphor-imaging system (Molecular Dynamics, Sunnyvale, CA).