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
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Mentions: To further characterize the DksA–iNTPs interplay, we reasoned that if the mechanism of action of DksA is to interfere with nucleotide binding, DksA should have little or no effect on RPo formed at the nucleotide-insensitive λ pR promoter. Therefore, experiments were conducted to investigate the effect of DksA on the large DNA compaction and high promoter occupancy observed at λ pR. For these experiments, RPo were assembled at pR using RNAP preincubated with 325 nM or 650 nM DksA. The DNA contour length measurements reveal that the DNA compaction is not affected by saturating concentrations of DksA as it remains at values around 30 nm (Figure 5A and B); likewise, the promoter occupancy is not significantly altered by 650 nM DksA, showing even a slight increase from 22.1% to 24.2% (Table 1). These results reinforce the hypothesis that DksA interferes with nucleotide positioning into the RNAP active site, thus affecting primarily iNTPs-sensitive promoters such as the ribosomal promoters.
Affiliation: Dipartimento di Bioscienze, Università degli Studi di Parma, Parma, Italy.