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A novel transcriptional regulator of L-arabinose utilization in human gut bacteria.

Chang C, Tesar C, Li X, Kim Y, Rodionov DA, Joachimiak A - Nucleic Acids Res. (2015)

Bottom Line: L-arabinose was confirmed to be a negative effector of BtAraR.In the structure of the BtAraR-DNA complex, we found the unique interaction of arginine intercalating its guanidinum moiety into the base pair stacking of B-DNA.L-arabinose binding induces movement of wHTH domains, resulting in a conformation unsuitable for DNA binding.

View Article: PubMed Central - PubMed

Affiliation: Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne, IL 60439, USA Structural Biology Center, Biosciences Division, Argonne National Laboratory, Argonne, IL 60439, USA.

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EMSA with BtAraR protein and its target DNA operators. (A–C) Titration of BtAraR protein for binding to DNA fragments from the BT0356 and BT0365 genes and the negative control (N.C.), which is a DNA fragment containing the shuffled sequence of BT0356. (D–E) Influence of sugar effectors on the formation of the BtAraR–DNA complex. The BtAraR protein (1 μM) and the BT0356 DNA fragment (0.2 nM) were incubated with increasing concentrations of L-arabinose and D-xylose in the incubation mixture (0.01–2 mM).
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Figure 2: EMSA with BtAraR protein and its target DNA operators. (A–C) Titration of BtAraR protein for binding to DNA fragments from the BT0356 and BT0365 genes and the negative control (N.C.), which is a DNA fragment containing the shuffled sequence of BT0356. (D–E) Influence of sugar effectors on the formation of the BtAraR–DNA complex. The BtAraR protein (1 μM) and the BT0356 DNA fragment (0.2 nM) were incubated with increasing concentrations of L-arabinose and D-xylose in the incubation mixture (0.01–2 mM).

Mentions: To validate the ability of BtAraR to specifically bind to the predicted DNA sites and to assess the role of possible effectors, we used EMSA. The BT0354 gene from B. thetaiotaomicron was cloned and overexpressed in E. coli and the recombinant BtAraR protein was purified to homogeneity. Binding of apo–BtAraR to synthetic biotin-labeled DNA fragments containing the predicted AraR sites at the AraR promoter regions of BT0356 and BT0365 genes was assessed. The 65-bp DNA fragment of the BT0356 promoter contains two tandem putative AraR sites, while the 41-bp DNA fragment of the BT0365 promoter represents a single AraR site. For both DNA fragments, mobility shifts of the DNA bands were observed in the presence of apo-BtAraR protein at concentrations of 1.0 μM (Figure 2A and B). For the BT0356 promoter two bands of protein/DNA complexes are observed, likely corresponding to one and two dimers of BtAraR bound to the promoter. The BT0365 promoter has a single AraR site and only one shifted band is observed. No gel shift of the DNA band was detected in the presence of control DNA (Figure 2C), confirming that apo-BtAraR recognizes specific DNA operator sequences.


A novel transcriptional regulator of L-arabinose utilization in human gut bacteria.

Chang C, Tesar C, Li X, Kim Y, Rodionov DA, Joachimiak A - Nucleic Acids Res. (2015)

EMSA with BtAraR protein and its target DNA operators. (A–C) Titration of BtAraR protein for binding to DNA fragments from the BT0356 and BT0365 genes and the negative control (N.C.), which is a DNA fragment containing the shuffled sequence of BT0356. (D–E) Influence of sugar effectors on the formation of the BtAraR–DNA complex. The BtAraR protein (1 μM) and the BT0356 DNA fragment (0.2 nM) were incubated with increasing concentrations of L-arabinose and D-xylose in the incubation mixture (0.01–2 mM).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4666351&req=5

Figure 2: EMSA with BtAraR protein and its target DNA operators. (A–C) Titration of BtAraR protein for binding to DNA fragments from the BT0356 and BT0365 genes and the negative control (N.C.), which is a DNA fragment containing the shuffled sequence of BT0356. (D–E) Influence of sugar effectors on the formation of the BtAraR–DNA complex. The BtAraR protein (1 μM) and the BT0356 DNA fragment (0.2 nM) were incubated with increasing concentrations of L-arabinose and D-xylose in the incubation mixture (0.01–2 mM).
Mentions: To validate the ability of BtAraR to specifically bind to the predicted DNA sites and to assess the role of possible effectors, we used EMSA. The BT0354 gene from B. thetaiotaomicron was cloned and overexpressed in E. coli and the recombinant BtAraR protein was purified to homogeneity. Binding of apo–BtAraR to synthetic biotin-labeled DNA fragments containing the predicted AraR sites at the AraR promoter regions of BT0356 and BT0365 genes was assessed. The 65-bp DNA fragment of the BT0356 promoter contains two tandem putative AraR sites, while the 41-bp DNA fragment of the BT0365 promoter represents a single AraR site. For both DNA fragments, mobility shifts of the DNA bands were observed in the presence of apo-BtAraR protein at concentrations of 1.0 μM (Figure 2A and B). For the BT0356 promoter two bands of protein/DNA complexes are observed, likely corresponding to one and two dimers of BtAraR bound to the promoter. The BT0365 promoter has a single AraR site and only one shifted band is observed. No gel shift of the DNA band was detected in the presence of control DNA (Figure 2C), confirming that apo-BtAraR recognizes specific DNA operator sequences.

Bottom Line: L-arabinose was confirmed to be a negative effector of BtAraR.In the structure of the BtAraR-DNA complex, we found the unique interaction of arginine intercalating its guanidinum moiety into the base pair stacking of B-DNA.L-arabinose binding induces movement of wHTH domains, resulting in a conformation unsuitable for DNA binding.

View Article: PubMed Central - PubMed

Affiliation: Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne, IL 60439, USA Structural Biology Center, Biosciences Division, Argonne National Laboratory, Argonne, IL 60439, USA.

Show MeSH