The NH2-terminal 108 amino acids of the Escherichia coli DnaJ protein stimulate the ATPase activity of DnaK and are sufficient for lambda replication.
The Escherichia coli heat shock proteins DnaK and DnaJ function cooperatively as molecular chaperones. Central to their biochemical functions is the ability of DnaJ to interact with DnaK and to stimulate its ATPase activity. Here, we report the genetic isolation of dnaJ12, which has a nonsense mutation at codon 109, yet was able to support lambda growth at 30 degrees C. The 12-kDa DnaJ12 protein was purified to homogeneity and shown to be active in an in vitro lambda-DNA replication system and to be capable of stimulating DnaK's ATPase activity, specifically at the step of ATP hydrolysis. The previously well studied and characterized dnaJ259 mutation was also cloned and sequenced, revealing a single His-->Gln amino acid change at codon 33. The purified DnaJ259 protein was inactive in an in vitro lambda-DNA replication system and was unable to stimulate DnaK's ATPase activity. Consistent with this, an NH2-terminal deletion of the first 34 amino acids or an Asp insertion at residue 35 of DnaJ resulted in a protein that completely lacked DnaJ activity. Collectively, these results demonstrate that the highly conserved NH2-terminal region of DnaJ, the so-called J region, is necessary and sufficient for stimulating both DnaK's ATPase activity and lambda-DNA replication. These results may be applicable to other eukaryotic proteins that contain this conserved J domain as proteins that interact and stimulate the hydrolysis of ATP by their cognate HSP70 proteins.
Wall
D
D
Department of Cellular, Viral, and Molecular Biology, University of Utah Medical Center, Salt Lake City 84132.
Zylicz
M
M
Georgopoulos
C
C
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D010582:Bacteriophage lambda / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D001483:Base Sequence; D004261:DNA Replication; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016296:Mutagenesis; D010446:Peptide Fragments / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D014357:Trypsin / Q000378:metabolism; D014779:Virus Replication; D016335:Zinc Fingers
D001426:Bacterial Proteins; C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D006898:Hydroxylamines; D010446:Peptide Fragments; D019811:Hydroxylamine; D014357:Trypsin; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli