PubMed:10406954 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PubMed/sourceid/10406954","sourcedb":"PubMed","sourceid":"10406954","source_url":"http://www.ncbi.nlm.nih.gov/pubmed/10406954","text":"Phosphorylation, dephosphorylation and DNA-binding of the Bradyrhizobium japonicum RegSR two-component regulatory proteins.\nUnder low oxygen conditions, induction of many genes required for nitrogen fixation in Bradyrhizobium japonicum depends on the redox-responsive transcriptional activator NifA which is encoded in the fixR-nifA operon. Basal expression of this operon depends on the response regulator RegR and a DNA element located around position -68 in the fixR-nifA promoter region. To investigate the functional properties of RegR and the interaction with its putative cognate kinase, RegS, we overproduced and affinity-purified RegR and a truncated soluble variant of RegS (RegS(C)), both as N-terminally His(6)-tagged proteins. RegS(C) autophosphorylated when incubated with [gamma-(32)P]ATP, and it catalyzed the transfer of the phosphoryl label to RegR. The phosphorylated form of RegS(C) exhibited phosphatase activity on RegR-phosphate. Chemical stability tests and site-specific mutagenesis identified amino acids H219 and D63 of RegS and RegR, respectively, as the phosphorylated residues. Competition experiments with isolated domains demonstrated that the N-terminal but not the C-terminal domain of RegR interacts with RegS(C). Band-shift experiments revealed that phosphorylated RegR had at least eightfold enhanced DNA-binding activity compared with dephosphorylated RegR or the mutant protein RegR-D63N, which cannot be phosphorylated. In conclusion, the RegSR proteins of B. japonicum exhibit functional properties in vitro that are typical of two-component regulatory systems.","tracks":[{"project":"FSU-PRGE","denotations":[{"id":"T1","span":{"begin":83,"end":88},"obj":"protein"},{"id":"T2","span":{"begin":294,"end":298},"obj":"protein"},{"id":"T3","span":{"begin":323,"end":339},"obj":"protein"},{"id":"T4","span":{"begin":407,"end":411},"obj":"protein"},{"id":"T5","span":{"begin":465,"end":469},"obj":"protein"},{"id":"T6","span":{"begin":470,"end":474},"obj":"protein"},{"id":"T7","span":{"begin":536,"end":540},"obj":"protein"},{"id":"T8","span":{"begin":587,"end":593},"obj":"protein"},{"id":"T9","span":{"begin":595,"end":599},"obj":"protein"},{"id":"T10","span":{"begin":639,"end":643},"obj":"protein"},{"id":"T11","span":{"begin":679,"end":683},"obj":"protein"},{"id":"T12","span":{"begin":685,"end":689},"obj":"protein"},{"id":"T13","span":{"begin":740,"end":744},"obj":"protein"},{"id":"T14","span":{"begin":862,"end":866},"obj":"protein"},{"id":"T15","span":{"begin":895,"end":899},"obj":"protein"},{"id":"T16","span":{"begin":913,"end":924},"obj":"protein"},{"id":"T17","span":{"begin":937,"end":941},"obj":"protein"},{"id":"T18","span":{"begin":1047,"end":1051},"obj":"protein"},{"id":"T19","span":{"begin":1056,"end":1060},"obj":"protein"},{"id":"T20","span":{"begin":1220,"end":1224},"obj":"protein"},{"id":"T21","span":{"begin":1240,"end":1244},"obj":"protein"},{"id":"T22","span":{"begin":1301,"end":1305},"obj":"protein"},{"id":"T23","span":{"begin":1390,"end":1394},"obj":"protein"},{"id":"T24","span":{"begin":1417,"end":1421},"obj":"protein"},{"id":"T25","span":{"begin":1479,"end":1484},"obj":"protein"}],"attributes":[{"subj":"T1","pred":"source","obj":"FSU-PRGE"},{"subj":"T2","pred":"source","obj":"FSU-PRGE"},{"subj":"T3","pred":"source","obj":"FSU-PRGE"},{"subj":"T4","pred":"source","obj":"FSU-PRGE"},{"subj":"T5","pred":"source","obj":"FSU-PRGE"},{"subj":"T6","pred":"source","obj":"FSU-PRGE"},{"subj":"T7","pred":"source","obj":"FSU-PRGE"},{"subj":"T8","pred":"source","obj":"FSU-PRGE"},{"subj":"T9","pred":"source","obj":"FSU-PRGE"},{"subj":"T10","pred":"source","obj":"FSU-PRGE"},{"subj":"T11","pred":"source","obj":"FSU-PRGE"},{"subj":"T12","pred":"source","obj":"FSU-PRGE"},{"subj":"T13","pred":"source","obj":"FSU-PRGE"},{"subj":"T14","pred":"source","obj":"FSU-PRGE"},{"subj":"T15","pred":"source","obj":"FSU-PRGE"},{"subj":"T16","pred":"source","obj":"FSU-PRGE"},{"subj":"T17","pred":"source","obj":"FSU-PRGE"},{"subj":"T18","pred":"source","obj":"FSU-PRGE"},{"subj":"T19","pred":"source","obj":"FSU-PRGE"},{"subj":"T20","pred":"source","obj":"FSU-PRGE"},{"subj":"T21","pred":"source","obj":"FSU-PRGE"},{"subj":"T22","pred":"source","obj":"FSU-PRGE"},{"subj":"T23","pred":"source","obj":"FSU-PRGE"},{"subj":"T24","pred":"source","obj":"FSU-PRGE"},{"subj":"T25","pred":"source","obj":"FSU-PRGE"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"FSU-PRGE","color":"#ec9396","default":true}]}]}}