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Structure and dynamics of polymyxin-resistance-associated response regulator PmrA in complex with promoter DNA.

Lou YC, Weng TH, Li YC, Kao YF, Lin WF, Peng HL, Chou SH, Hsiao CD, Chen C - Nat Commun (2015)

Bottom Line: However, NMR studies show that in the DNA-bound state, two domains tumble separately and an REC-DBD interaction is transiently populated in solution.Reporter gene analyses of PmrA variants with altered interface residues suggest that the interface is not crucial for supporting gene expression.We propose that REC-DBD interdomain dynamics and the DBD-DBD interface help PmrA interact with RNA polymerase holoenzyme to activate downstream gene transcription.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, ROC.

ABSTRACT
PmrA, an OmpR/PhoB family response regulator, manages genes for antibiotic resistance. Phosphorylation of OmpR/PhoB response regulator induces the formation of a symmetric dimer in the N-terminal receiver domain (REC), promoting two C-terminal DNA-binding domains (DBDs) to recognize promoter DNA to elicit adaptive responses. Recently, determination of the KdpE-DNA complex structure revealed an REC-DBD interface in the upstream protomer that may be necessary for transcription activation. Here, we report the 3.2-Å-resolution crystal structure of the PmrA-DNA complex, which reveals a similar yet different REC-DBD interface. However, NMR studies show that in the DNA-bound state, two domains tumble separately and an REC-DBD interaction is transiently populated in solution. Reporter gene analyses of PmrA variants with altered interface residues suggest that the interface is not crucial for supporting gene expression. We propose that REC-DBD interdomain dynamics and the DBD-DBD interface help PmrA interact with RNA polymerase holoenzyme to activate downstream gene transcription.

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Promoter recognition and transcription activity.(a) The bindings between PmrA, WT-PmrA or its variants and DNA measured by fluorescence polarization experiments are fitted by a one-site binding model. See also Supplementary Fig. 11 for binding affinities. (b) β-Galactosidase reporter assay in K. pneumoniae carrying the plasmid that expresses PmrA, WT-PmrA or its variants and the reporter plasmid that contains the pbgP promoter in front of the lacZ gene. The expression of PmrA, WT-PmrA or its variants was induced by isopropyl-β-D-thiogalactoside (IPTG, 1 mM ml−1) until the cells grew to the mid-logarithmic phase. The β-galactosidase assay in cells that carry WT-PmrA plasmid but without the addition of IPTG is denoted non-WT, which represents the production of β-galactosidase induced by endogenous PmrA. Reporter assay results are expressed as Miller Units. All experiments were performed in triplicate. Error bars are defined as s.d.
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f7: Promoter recognition and transcription activity.(a) The bindings between PmrA, WT-PmrA or its variants and DNA measured by fluorescence polarization experiments are fitted by a one-site binding model. See also Supplementary Fig. 11 for binding affinities. (b) β-Galactosidase reporter assay in K. pneumoniae carrying the plasmid that expresses PmrA, WT-PmrA or its variants and the reporter plasmid that contains the pbgP promoter in front of the lacZ gene. The expression of PmrA, WT-PmrA or its variants was induced by isopropyl-β-D-thiogalactoside (IPTG, 1 mM ml−1) until the cells grew to the mid-logarithmic phase. The β-galactosidase assay in cells that carry WT-PmrA plasmid but without the addition of IPTG is denoted non-WT, which represents the production of β-galactosidase induced by endogenous PmrA. Reporter assay results are expressed as Miller Units. All experiments were performed in triplicate. Error bars are defined as s.d.

Mentions: To establish whether PmrA is biologically active and the formation of extensive REC–DBD interface is important for promoter recognition and transcription activation, we performed in vitro fluorescence polarization experiments (Fig. 7a and Supplementary Fig. 12) to measure the binding affinities between PmrA, WT-PmrA or its variants, and fluorescence-labelled DNA as well as in vivo β-galactosidase reporter assay (Fig. 7b) to monitor the transcription activity of all protein constructs. PmrA (the double-substitution W181G/I220D variant) retains full activity in promotor recognition (Kd values for WT-PmrA and PmrA to DNA of 193.5±7.7 and 200.6±8.2 nM, respectively) and transcription activation as for WT-PmrA. The residue Trp181 is in the N terminus of the transactivation loop, and the single-mutant W181G exhibits slightly lower transcript activity than that of WT-PmrA. The replacement of residues involved in DNA recognition by alanine reduces the DNA-binding affinity significantly (Kd values for N188A, N196A and R210A to DNA of 762.2±12.3, 398.9±10.9 and 3036.8±11.7 nM, respectively) and hence abolishes the activity in transcription. The mutants that change residues in the REC–DBD interface mostly exhibit a comparable DNA-binding affinity to that of WT-PmrA (Supplementary Fig. 12), with the exception of N176A (Kd=364.9±11.6 nM), which therefore reveals decreased activity in transcription. Interestingly, induced expression of R160A leads to a significant increase (∼2.7-fold) in transcription, but the production of N43A, S46A and N120A results in slight-to-moderately reduced transcription. Western blot analysis (Supplementary Fig. 13) shows that the expression of most of the protein constructs in K. pneumoniae are equivalent to that of WT-PmrA, but N43A and S46A show decreased expression, which suggests that the β-galactosidase activity of both mutants can be higher if their expression levels resemble that of WT-PmrA. The alterations in interface residues mostly do not significantly interfere with their activities in promoter recognition and transcription activation. These analyses show that PmrA is biologically active and the formation of an REC–DBD interface is not crucial for activating downstream gene transcription.


Structure and dynamics of polymyxin-resistance-associated response regulator PmrA in complex with promoter DNA.

Lou YC, Weng TH, Li YC, Kao YF, Lin WF, Peng HL, Chou SH, Hsiao CD, Chen C - Nat Commun (2015)

Promoter recognition and transcription activity.(a) The bindings between PmrA, WT-PmrA or its variants and DNA measured by fluorescence polarization experiments are fitted by a one-site binding model. See also Supplementary Fig. 11 for binding affinities. (b) β-Galactosidase reporter assay in K. pneumoniae carrying the plasmid that expresses PmrA, WT-PmrA or its variants and the reporter plasmid that contains the pbgP promoter in front of the lacZ gene. The expression of PmrA, WT-PmrA or its variants was induced by isopropyl-β-D-thiogalactoside (IPTG, 1 mM ml−1) until the cells grew to the mid-logarithmic phase. The β-galactosidase assay in cells that carry WT-PmrA plasmid but without the addition of IPTG is denoted non-WT, which represents the production of β-galactosidase induced by endogenous PmrA. Reporter assay results are expressed as Miller Units. All experiments were performed in triplicate. Error bars are defined as s.d.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4660055&req=5

f7: Promoter recognition and transcription activity.(a) The bindings between PmrA, WT-PmrA or its variants and DNA measured by fluorescence polarization experiments are fitted by a one-site binding model. See also Supplementary Fig. 11 for binding affinities. (b) β-Galactosidase reporter assay in K. pneumoniae carrying the plasmid that expresses PmrA, WT-PmrA or its variants and the reporter plasmid that contains the pbgP promoter in front of the lacZ gene. The expression of PmrA, WT-PmrA or its variants was induced by isopropyl-β-D-thiogalactoside (IPTG, 1 mM ml−1) until the cells grew to the mid-logarithmic phase. The β-galactosidase assay in cells that carry WT-PmrA plasmid but without the addition of IPTG is denoted non-WT, which represents the production of β-galactosidase induced by endogenous PmrA. Reporter assay results are expressed as Miller Units. All experiments were performed in triplicate. Error bars are defined as s.d.
Mentions: To establish whether PmrA is biologically active and the formation of extensive REC–DBD interface is important for promoter recognition and transcription activation, we performed in vitro fluorescence polarization experiments (Fig. 7a and Supplementary Fig. 12) to measure the binding affinities between PmrA, WT-PmrA or its variants, and fluorescence-labelled DNA as well as in vivo β-galactosidase reporter assay (Fig. 7b) to monitor the transcription activity of all protein constructs. PmrA (the double-substitution W181G/I220D variant) retains full activity in promotor recognition (Kd values for WT-PmrA and PmrA to DNA of 193.5±7.7 and 200.6±8.2 nM, respectively) and transcription activation as for WT-PmrA. The residue Trp181 is in the N terminus of the transactivation loop, and the single-mutant W181G exhibits slightly lower transcript activity than that of WT-PmrA. The replacement of residues involved in DNA recognition by alanine reduces the DNA-binding affinity significantly (Kd values for N188A, N196A and R210A to DNA of 762.2±12.3, 398.9±10.9 and 3036.8±11.7 nM, respectively) and hence abolishes the activity in transcription. The mutants that change residues in the REC–DBD interface mostly exhibit a comparable DNA-binding affinity to that of WT-PmrA (Supplementary Fig. 12), with the exception of N176A (Kd=364.9±11.6 nM), which therefore reveals decreased activity in transcription. Interestingly, induced expression of R160A leads to a significant increase (∼2.7-fold) in transcription, but the production of N43A, S46A and N120A results in slight-to-moderately reduced transcription. Western blot analysis (Supplementary Fig. 13) shows that the expression of most of the protein constructs in K. pneumoniae are equivalent to that of WT-PmrA, but N43A and S46A show decreased expression, which suggests that the β-galactosidase activity of both mutants can be higher if their expression levels resemble that of WT-PmrA. The alterations in interface residues mostly do not significantly interfere with their activities in promoter recognition and transcription activation. These analyses show that PmrA is biologically active and the formation of an REC–DBD interface is not crucial for activating downstream gene transcription.

Bottom Line: However, NMR studies show that in the DNA-bound state, two domains tumble separately and an REC-DBD interaction is transiently populated in solution.Reporter gene analyses of PmrA variants with altered interface residues suggest that the interface is not crucial for supporting gene expression.We propose that REC-DBD interdomain dynamics and the DBD-DBD interface help PmrA interact with RNA polymerase holoenzyme to activate downstream gene transcription.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, ROC.

ABSTRACT
PmrA, an OmpR/PhoB family response regulator, manages genes for antibiotic resistance. Phosphorylation of OmpR/PhoB response regulator induces the formation of a symmetric dimer in the N-terminal receiver domain (REC), promoting two C-terminal DNA-binding domains (DBDs) to recognize promoter DNA to elicit adaptive responses. Recently, determination of the KdpE-DNA complex structure revealed an REC-DBD interface in the upstream protomer that may be necessary for transcription activation. Here, we report the 3.2-Å-resolution crystal structure of the PmrA-DNA complex, which reveals a similar yet different REC-DBD interface. However, NMR studies show that in the DNA-bound state, two domains tumble separately and an REC-DBD interaction is transiently populated in solution. Reporter gene analyses of PmrA variants with altered interface residues suggest that the interface is not crucial for supporting gene expression. We propose that REC-DBD interdomain dynamics and the DBD-DBD interface help PmrA interact with RNA polymerase holoenzyme to activate downstream gene transcription.

Show MeSH
Related in: MedlinePlus