Sequential recognition of two distinct sites in sigma(S) by the proteolytic targeting factor RssB and ClpX.
sigma(S) (RpoS), the master regulator of the general stress response in Escherichia coli, is a model system for regulated proteolysis in bacteria. sigma(S) turnover requires ClpXP and the response regulator RssB, whose phosphorylated form exhibits high affinity for sigma(S). Here, we demonstrate that recognition by the RssB/ClpXP system involves two distinct regions in sigma(S). Region 2.5 of sigma(S) (a long alpha-helix) is sufficient for binding of phosphorylated RssB. However, this interaction alone is not sufficient to trigger proteolysis. A second region located in the N-terminal part of sigma(S), which is exposed only upon RssB-sigma(S) interaction, serves as a binding site for the ClpX chaperone. Binding of the ClpX hexameric ring to sigma(S)-derived reporter proteins carrying the ClpX-binding site (but not the RssB-binding site) is also not sufficient to commit the protein to degradation. Our data indicate that RssB plays a second role in the initiation of sigma(S) proteolysis that goes beyond targeting of sigma(S) to ClpX, and suggest a model for the sequence of events in the initiation of sigma(S) proteolysis.
Stüdemann
Andrea
A
Institut für Biologie-Mikrobiologie, Freie Universität Berlin, D-14195 Berlin, Germany.
Noirclerc-Savoye
Marjolaine
M
Klauck
Eberhard
E
Becker
Gisela
G
Schneider
Dominique
D
Hengge
Regine
R
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000409:Alanine / Q000378:metabolism; D019943:Amino Acid Substitution; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017354:Point Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012808:Sigma Factor / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; D012808:Sigma Factor; D014157:Transcription Factors; C098923:rssB protein, E coli; D000251:Adenosine Triphosphatases; D000409:Alanine