New insights into the regulatory mechanisms of ppGpp and DksA on Escherichia coli RNA polymerase-promoter complex.
Bottom Line: Addition of the modulators affects both DNA wrapping and RPo stability in a promoter-dependent manner.Overall, the results obtained under different conditions of ppGpp, DksA and initiating nucleotides (iNTPs) indicate that ppGpp allosterically prevents the conformational changes associated with an extended DNA wrapping that leads to RPo stabilization, while DksA interferes directly with nucleotide positioning into the RNAP active site.At the iNTPs-sensitive rRNA promoters ppGpp and DksA display an independent inhibitory effect, while at the iNTPs-insensitive pR promoter DksA reduces the effect of ppGpp in accordance with their antagonistic role.
Affiliation: Dipartimento di Bioscienze, Università degli Studi di Parma, Parma, Italy.Show MeSH
Related in: MedlinePlus
Mentions: Transcription reactions were assembled in 10 μl final volume as follows: RNAP (35 nM) was preincubated in transcription buffer with none, one or both modulators at the concentrations indicated in Figure 8, for 10 min at 37°C. Afterward, DNA templates (20 nM) and iNTPs (as indicated in Figure 8) were added and the reaction was incubated for 15 min at 37°C to favor RPo formation. Transcription was started by the addition of 1 μl NTP mix composed of 200 μM ATP, CTP and GTP, 100 μM UTP and 1 mg/ml heparin immediately after the addition of [α-32P]UTP (1 μCi; 1 Ci = 37 GBq). After 10 min of incubation at 30°C the reaction was terminated by the addition of 10 μl of formamide loading buffer. Electrophoresis was performed on a 7 M urea-6% polyacrylamide gel and the RNA transcript was quantified by a Packard Cyclone (Alliance Analytical). The in vitro transcription assays were repeated at least two times.
Affiliation: Dipartimento di Bioscienze, Università degli Studi di Parma, Parma, Italy.