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A new glimpse of FadR-DNA crosstalk revealed by deep dissection of the E. coli FadR regulatory protein.

Zhang Y, Gao R, Ye H, Wang Q, Feng Y - Protein Cell (2014)

Bottom Line: We also observed that a single genetically-recessive mutation of W60G in FadR regulatory protein can lead to loss of its DNA-binding activity, and thereby impair all the regulatory roles in fatty acid metabolisms.Structural analyses of FadR protein indicated that the hydrophobic interaction amongst the three amino acids (W60, F74 and W75) is critical for its DNA-binding ability by maintaining the configuration of its neighboring two β-sheets.Further site-directed mutagenesis analyses demonstrated that the FadR mutants (F74G and/or W75G) do not exhibit the detected DNA-binding activity, validating above structural reasoning.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Nanjing Normal University, Nanjing, 210023, China.

ABSTRACT
Escherichia coli (E. coli) FadR regulator plays dual roles in fatty acid metabolism, which not only represses the fatty acid degradation (fad) system, but also activates the unsaturated fatty acid synthesis pathway. Earlier structural and biochemical studies of FadR protein have provided insights into interplay between FadR protein with its DNA target and/or ligand, while the missing knowledge gap (esp. residues with indirect roles in DNA binding) remains unclear. Here we report this case through deep mapping of old E. coli fadR mutants accumulated. Molecular dissection of E. coli K113 strain, a fadR mutant that can grow on decanoic acid (C10) as sole carbon sources unexpectedly revealed a single point mutation of T178G in fadR locus (W60G in FadRk113). We also observed that a single genetically-recessive mutation of W60G in FadR regulatory protein can lead to loss of its DNA-binding activity, and thereby impair all the regulatory roles in fatty acid metabolisms. Structural analyses of FadR protein indicated that the hydrophobic interaction amongst the three amino acids (W60, F74 and W75) is critical for its DNA-binding ability by maintaining the configuration of its neighboring two β-sheets. Further site-directed mutagenesis analyses demonstrated that the FadR mutants (F74G and/or W75G) do not exhibit the detected DNA-binding activity, validating above structural reasoning.

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Mutant FadR protein (FadRK113) losses its DNA binding activityin vitro. The two FadR-binding sites from both fadL (A) or fadD (B) can bind to the wild-type FadR protein (FadRWT), whereas not the mutant version of FadR, FadRk113 (W60G). (C) The wild-type FadR protein (FadRWT) can bind to promoter regions covering the FadR-binding sites of fadBA and fadM, whereas FadRk113 does not. (D) FadRk113 fails to bind to promoter regions of fabA and fabB, two UFA biosynthetic genes whereas FadRWT does. All the EMSA experiments were carried out using 7% native PAGE, and a representative result is shown here. In gel shift assays, FadRWT is added as follows: 0, 0.5 and 1 pmol. Similarly, FadRK113 is supplemented in 0, 0.5, 1 and 2 pmol, respectively. All the DIG-labeled probes are added to 0.1 pmol
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Fig3: Mutant FadR protein (FadRK113) losses its DNA binding activityin vitro. The two FadR-binding sites from both fadL (A) or fadD (B) can bind to the wild-type FadR protein (FadRWT), whereas not the mutant version of FadR, FadRk113 (W60G). (C) The wild-type FadR protein (FadRWT) can bind to promoter regions covering the FadR-binding sites of fadBA and fadM, whereas FadRk113 does not. (D) FadRk113 fails to bind to promoter regions of fabA and fabB, two UFA biosynthetic genes whereas FadRWT does. All the EMSA experiments were carried out using 7% native PAGE, and a representative result is shown here. In gel shift assays, FadRWT is added as follows: 0, 0.5 and 1 pmol. Similarly, FadRK113 is supplemented in 0, 0.5, 1 and 2 pmol, respectively. All the DIG-labeled probes are added to 0.1 pmol

Mentions: To fully address the effect of the genetically-recessive single mutation of W60G on regulatory function of FadR protein, we employed the in vitro and in vivo approaches including EMSA and transcriptional fusion. The DNA binding activity of the mutant FadR protein (FadRK113) was examined in vitro using gel shift assays. Eight sets of DIG-labeled DNA probes were prepared. Among them, two probes were designed for either fadL or fadD in that they have two FadR-binding palindromes (fadL probe1 plus fadL probe 2 (Fig. 3A) and fadD probe1 plus fadD probe 2 (Fig. 3B)) (Feng & Cronan, 2012). As we expected, the wild-type FadR protein (FadRWT) exhibited obvious binding to fadL (fadD) promoter (Fig. 3A and 3B), however the FadRK113 protein was not visualized to interact with them at the same protein level used in our trials (no more than 10 pmol). The promoters of fadBA (Feng & Cronan, 2012) and fadM (Feng & Cronan, 2009b), two members of β-oxidation system, also cannot be bound by FadRK113 protein (Fig. 3C). The similar scenario was also observed with fabA and fabB (Feng & Cronan, 2011a), two important genes required for UFA biosynthetic pathway (Fig. 3D).Figure 3


A new glimpse of FadR-DNA crosstalk revealed by deep dissection of the E. coli FadR regulatory protein.

Zhang Y, Gao R, Ye H, Wang Q, Feng Y - Protein Cell (2014)

Mutant FadR protein (FadRK113) losses its DNA binding activityin vitro. The two FadR-binding sites from both fadL (A) or fadD (B) can bind to the wild-type FadR protein (FadRWT), whereas not the mutant version of FadR, FadRk113 (W60G). (C) The wild-type FadR protein (FadRWT) can bind to promoter regions covering the FadR-binding sites of fadBA and fadM, whereas FadRk113 does not. (D) FadRk113 fails to bind to promoter regions of fabA and fabB, two UFA biosynthetic genes whereas FadRWT does. All the EMSA experiments were carried out using 7% native PAGE, and a representative result is shown here. In gel shift assays, FadRWT is added as follows: 0, 0.5 and 1 pmol. Similarly, FadRK113 is supplemented in 0, 0.5, 1 and 2 pmol, respectively. All the DIG-labeled probes are added to 0.1 pmol
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig3: Mutant FadR protein (FadRK113) losses its DNA binding activityin vitro. The two FadR-binding sites from both fadL (A) or fadD (B) can bind to the wild-type FadR protein (FadRWT), whereas not the mutant version of FadR, FadRk113 (W60G). (C) The wild-type FadR protein (FadRWT) can bind to promoter regions covering the FadR-binding sites of fadBA and fadM, whereas FadRk113 does not. (D) FadRk113 fails to bind to promoter regions of fabA and fabB, two UFA biosynthetic genes whereas FadRWT does. All the EMSA experiments were carried out using 7% native PAGE, and a representative result is shown here. In gel shift assays, FadRWT is added as follows: 0, 0.5 and 1 pmol. Similarly, FadRK113 is supplemented in 0, 0.5, 1 and 2 pmol, respectively. All the DIG-labeled probes are added to 0.1 pmol
Mentions: To fully address the effect of the genetically-recessive single mutation of W60G on regulatory function of FadR protein, we employed the in vitro and in vivo approaches including EMSA and transcriptional fusion. The DNA binding activity of the mutant FadR protein (FadRK113) was examined in vitro using gel shift assays. Eight sets of DIG-labeled DNA probes were prepared. Among them, two probes were designed for either fadL or fadD in that they have two FadR-binding palindromes (fadL probe1 plus fadL probe 2 (Fig. 3A) and fadD probe1 plus fadD probe 2 (Fig. 3B)) (Feng & Cronan, 2012). As we expected, the wild-type FadR protein (FadRWT) exhibited obvious binding to fadL (fadD) promoter (Fig. 3A and 3B), however the FadRK113 protein was not visualized to interact with them at the same protein level used in our trials (no more than 10 pmol). The promoters of fadBA (Feng & Cronan, 2012) and fadM (Feng & Cronan, 2009b), two members of β-oxidation system, also cannot be bound by FadRK113 protein (Fig. 3C). The similar scenario was also observed with fabA and fabB (Feng & Cronan, 2011a), two important genes required for UFA biosynthetic pathway (Fig. 3D).Figure 3

Bottom Line: We also observed that a single genetically-recessive mutation of W60G in FadR regulatory protein can lead to loss of its DNA-binding activity, and thereby impair all the regulatory roles in fatty acid metabolisms.Structural analyses of FadR protein indicated that the hydrophobic interaction amongst the three amino acids (W60, F74 and W75) is critical for its DNA-binding ability by maintaining the configuration of its neighboring two β-sheets.Further site-directed mutagenesis analyses demonstrated that the FadR mutants (F74G and/or W75G) do not exhibit the detected DNA-binding activity, validating above structural reasoning.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Nanjing Normal University, Nanjing, 210023, China.

ABSTRACT
Escherichia coli (E. coli) FadR regulator plays dual roles in fatty acid metabolism, which not only represses the fatty acid degradation (fad) system, but also activates the unsaturated fatty acid synthesis pathway. Earlier structural and biochemical studies of FadR protein have provided insights into interplay between FadR protein with its DNA target and/or ligand, while the missing knowledge gap (esp. residues with indirect roles in DNA binding) remains unclear. Here we report this case through deep mapping of old E. coli fadR mutants accumulated. Molecular dissection of E. coli K113 strain, a fadR mutant that can grow on decanoic acid (C10) as sole carbon sources unexpectedly revealed a single point mutation of T178G in fadR locus (W60G in FadRk113). We also observed that a single genetically-recessive mutation of W60G in FadR regulatory protein can lead to loss of its DNA-binding activity, and thereby impair all the regulatory roles in fatty acid metabolisms. Structural analyses of FadR protein indicated that the hydrophobic interaction amongst the three amino acids (W60, F74 and W75) is critical for its DNA-binding ability by maintaining the configuration of its neighboring two β-sheets. Further site-directed mutagenesis analyses demonstrated that the FadR mutants (F74G and/or W75G) do not exhibit the detected DNA-binding activity, validating above structural reasoning.

Show MeSH
Related in: MedlinePlus