THE NH2-TERMINAL 108 AMINO-ACIDS OF THE ESCHERICHIA-COLI DNAJ PROTEINSTIMULATE THE ATPASE ACTIVITY OF DNAK AND ARE SUFFICIENT FOR LAMBDA-REPLICATION

The Escherichia coil heat shock proteins DnaK and DnaJ function cooper atively as molecular chaperones. Central to their biochemical function s is the ability of DnaJ to interact with DnaK and to stimulate its AT Pase activity. Here, we report the genetic isolation of dnaJ12, which has a nonsense mutation at codon 109, yet was able to support lambda g rowth at 30 degrees C. The 12-kDa DnaJ12 protein was purified to homog eneity and shown to be active in an in vitro lambda-DNA replication sy stem and to be capable of stimulating DnaK's ATPase activity, specific ally at the step of ATP hydrolysis. The previously web studied and cha racterized dnaJ259 mutation was also cloned and sequenced, revealing a single His --> Gln amino acid change at codon 33. The purified DnaJ25 9 protein was inactive in an in vitro lambda-DNA replication system an d was unable to stimulate DnaK's ATPase activity. Consistent with this , an NH2-terminal deletion of the first 34 amino acids or an Asp inser tion at residue 35 of DnaJ resulted in a protein that completely lacke d DnaJ activity. Collectively, these results demonstrate that the high ly conserved NH2-terminal region of DnaJ, the so-called J region, is n ecessary and sufficient for stimulating both DnaK's ATPase activity an d lambda-DNA replication. These results may be applicable to other euk aryotic proteins that contain this conserved J domain as proteins that interact and stimulate the hydrolysis of ATP by their cognate HSP70 p roteins.