Interaction of the DnaK and DnaJ chaperone system with a native substrate, P1 RepA.
DnaK, the Hsp70 chaperone of Escherichia coli interacts with protein substrates in an ATP-dependent manner, in conjunction with DnaJ and GrpE co-chaperones, to carry out protein folding, protein remodeling, and assembly and disassembly of multisubunit protein complexes. To understand how DnaJ targets specific proteins for recognition by the DnaK chaperone system, we investigated the interaction of DnaJ and DnaK with a known natural substrate, bacteriophage P1 RepA protein. By characterizing RepA deletion derivatives, we found that DnaJ interacts with a region of RepA located between amino acids 180 and 200 of the 286-amino acid protein. A peptide corresponding to amino acids 180-195 inhibited the interaction of RepA and DnaJ. Two site-directed RepA mutants with alanine substitutions in this region were about 4-fold less efficiently activated for oriP1 DNA binding by DnaJ and DnaK than wild type RepA. We also identified by deletion analysis a site in RepA, in the region of amino acids 35-49, which interacts with DnaK. An alanine substitution mutant in amino acids 36-39 was constructed and found defective in activation by DnaJ and DnaK. Taken together the results suggest that DnaJ and DnaK interact with separate sites on RepA.
Kim
Soon-Young
SY
Laboratory of Molecular Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892-4264, USA.
Sharma
Suveena
S
Hoskins
Joel R
JR
Wickner
Sue
S
D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases; D004261:DNA Replication / Q000502:physiology; D004268:DNA-Binding Proteins / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism*; D018832:Molecular Chaperones / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D018741:Replication Origin; D015534:Trans-Activators
D004268:DNA-Binding Proteins; C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D010455:Peptides; D011506:Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D004247:DNA; C063842:dnaK protein, E coli; D004265:DNA Helicases; D000409:Alanine