Molecular analysis of the Rhodobacter capsulatus chaperone dnaKJ operon: purification and characterization of DnaK

Abstract In Rhodobacter capsulatus ( Rbc ), the participation of DnaK in the synthesis of light harvesting antenna complex I (LHI) has been recently inferred from the finding that the amount of LHI α- and β-polypeptides synthesized in an in vitro translation system was strongly reduced when DnaK was depleted. In the present work, a DnaK protein was isolated from Rbc and biochemically characterized. The N-terminus of the protein was sequenced and a corresponding oligo was used as probe in order to clone the gene coding for DnaK. The dnaK gene was located in an operon ( dnaKJ ) with two open reading frames, which code for DnaK and DnaJ, respectively. A promoter element corresponding to the consensus sequence of the atypical heat shock (HS) promoter of several α-purple proteobacteria was identified. Northern blot analysis indicated that dnaK and dnaJ belong to the same transcriptional unit; there were two transcripts, one comprising both the dnaK and dnaJ genes and a second with only dnaK . Primer extension analysis revealed that under both chemotrophic and phototrophic conditions transcription was initiated from the same position before and after HS. The promoter activity was studied under different growth conditions with a dnaK - lacZ fusion under the control of the dnaKJ promoter. The present work opens up the possibility to study the specific role of DnaK in the assembly of photosynthetic apparatus proteins.