Conformation of a plasmid replication initiator protein affects its proteolysis by ClpXP system.
Proteins from the Rep family of DNA replication initiators exist mainly as dimers, but only monomers can initiate DNA replication by interaction with the replication origin (ori). In this study, we investigated both the activation (monomerization) and the degradation of the broad-host-range plasmid RK2 replication initiation protein TrfA, which we found to be a member of a class of DNA replication initiators containing winged helix (WH) domains. Our in vivo and in vitro experiments demonstrated that the ClpX-dependent activation of TrfA leading to replicationally active protein monomers and mutations affecting TrfA dimer formation, result in the inhibition of TrfA protein degradation by the ClpXP proteolytic system. These data revealed that the TrfA monomers and dimers are degraded at substantially different rates. Our data also show that the plasmid replication initiator activity and stability in E. coli cells are affected by ClpXP system only when the protein sustains dimeric form.
Pierechod
Marcin
M
Department of Molecular and Cellular Biology, Faculty of Biotechnology, University of Gdansk, Gdansk, Poland.
Nowak
Agnieszka
A
Saari
Anna
A
Purta
Elzbieta
E
Bujnicki
Janusz M
JM
Konieczny
Igor
I
D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D019943:Amino Acid Substitution; D004261:DNA Replication; D049071:Endopeptidase Clp / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization / Q000235:genetics; D055550:Protein Stability
D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; C067405:TrfA protein, E coli; C483573:ClpXP protease, E coli; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C088072:ClpX protein, E coli; D000074183:ATPases Associated with Diverse Cellular Activities