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New insights into the regulatory mechanisms of ppGpp and DksA on Escherichia coli RNA polymerase-promoter complex.

Doniselli N, Rodriguez-Aliaga P, Amidani D, Bardales JA, Bustamante C, Guerra DG, Rivetti C - Nucleic Acids Res. (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Bioscienze, Università degli Studi di Parma, Parma, Italy.

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(A) A promoter complex with a manually traced outline (dashed line). (B) The same complex as in C with the sequence of pixels used for DNA contour length measurements highlighted in black. (C) Gallery of promoter complexes formed in the absence of modulators. (D) Gallery of promoter complexes formed with 200 μM ppGpp and 650 nM DksA. The broadening effect of the AFM tip does not allow to detect the presence of the modulators bound to the RNAP. Scale bars 100 nm.
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Figure 1: (A) A promoter complex with a manually traced outline (dashed line). (B) The same complex as in C with the sequence of pixels used for DNA contour length measurements highlighted in black. (C) Gallery of promoter complexes formed in the absence of modulators. (D) Gallery of promoter complexes formed with 200 μM ppGpp and 650 nM DksA. The broadening effect of the AFM tip does not allow to detect the presence of the modulators bound to the RNAP. Scale bars 100 nm.

Mentions: DNA contour length measurements were performed as detailed in the Supplementary data. Briefly, the DNA trace was digitized using custom-made Matlab scripts, by picking with the mouse several points along the DNA contour from one end to the other (Figure 1A). This digitized DNA trace served as an outline to identify the subset of pixels with higher intensity within a five pixel wide window, representing the DNA backbone. Next, binary images obtained by setting to 1 the DNA backbone pixels and to 0 the background were skeletonized with the bimorph built-in function of Matlab to generate eight-connected chaincode of unit thickness (Figure 1B). The DNA contour length was then determined by (ne, no) characterization using the following contour length estimator (32):\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{upgreek}\usepackage{mathrsfs}\setlength{\oddsidemargin}{-69pt}\begin{document}}{}\begin{equation*}L_{{\rm DNA}} = \left( {0.963n_e + 1.362n_o } \right) \times S/W\end{equation*}\end{document}where ne and no are the number of even and odd chain pixel respectively, S is the image scan size (2000 nm), W is the image width (512 pixels). An even chain element indicates a vertical or horizontal connection between two backbone pixels, while an odd chain element indicates a diagonal connection. Data were processed with MATLAB and graphed with Sigmaplot (Systat Software, Inc., California, USA). Histograms of all the DNA contour length distributions were constructed with a bin size of 5 nm. The resulting distributions passed the Shapiro–Wilk normality test of Sigmaplot and were therefore fitted to a Gaussian function.


New insights into the regulatory mechanisms of ppGpp and DksA on Escherichia coli RNA polymerase-promoter complex.

Doniselli N, Rodriguez-Aliaga P, Amidani D, Bardales JA, Bustamante C, Guerra DG, Rivetti C - Nucleic Acids Res. (2015)

(A) A promoter complex with a manually traced outline (dashed line). (B) The same complex as in C with the sequence of pixels used for DNA contour length measurements highlighted in black. (C) Gallery of promoter complexes formed in the absence of modulators. (D) Gallery of promoter complexes formed with 200 μM ppGpp and 650 nM DksA. The broadening effect of the AFM tip does not allow to detect the presence of the modulators bound to the RNAP. Scale bars 100 nm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4446441&req=5

Figure 1: (A) A promoter complex with a manually traced outline (dashed line). (B) The same complex as in C with the sequence of pixels used for DNA contour length measurements highlighted in black. (C) Gallery of promoter complexes formed in the absence of modulators. (D) Gallery of promoter complexes formed with 200 μM ppGpp and 650 nM DksA. The broadening effect of the AFM tip does not allow to detect the presence of the modulators bound to the RNAP. Scale bars 100 nm.
Mentions: DNA contour length measurements were performed as detailed in the Supplementary data. Briefly, the DNA trace was digitized using custom-made Matlab scripts, by picking with the mouse several points along the DNA contour from one end to the other (Figure 1A). This digitized DNA trace served as an outline to identify the subset of pixels with higher intensity within a five pixel wide window, representing the DNA backbone. Next, binary images obtained by setting to 1 the DNA backbone pixels and to 0 the background were skeletonized with the bimorph built-in function of Matlab to generate eight-connected chaincode of unit thickness (Figure 1B). The DNA contour length was then determined by (ne, no) characterization using the following contour length estimator (32):\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{upgreek}\usepackage{mathrsfs}\setlength{\oddsidemargin}{-69pt}\begin{document}}{}\begin{equation*}L_{{\rm DNA}} = \left( {0.963n_e + 1.362n_o } \right) \times S/W\end{equation*}\end{document}where ne and no are the number of even and odd chain pixel respectively, S is the image scan size (2000 nm), W is the image width (512 pixels). An even chain element indicates a vertical or horizontal connection between two backbone pixels, while an odd chain element indicates a diagonal connection. Data were processed with MATLAB and graphed with Sigmaplot (Systat Software, Inc., California, USA). Histograms of all the DNA contour length distributions were constructed with a bin size of 5 nm. The resulting distributions passed the Shapiro–Wilk normality test of Sigmaplot and were therefore fitted to a Gaussian function.

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.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Bioscienze, Università degli Studi di Parma, Parma, Italy.

Show MeSH
Related in: MedlinePlus