RESUMEN
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 alpha- and beta-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 alpha-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.
Asunto(s)
Proteínas de Escherichia coli , Proteínas HSP70 de Choque Térmico/genética , Chaperonas Moleculares/genética , Rhodobacter capsulatus/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Proteínas del Choque Térmico HSP40 , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Datos de Secuencia Molecular , Operón , ARN Bacteriano/genética , ARN Mensajero/genética , Mapeo RestrictivoRESUMEN
The radC gene, whose product plays a role in the prokaryotic repair of DNA damage after UV and X-ray irradiation, was cloned and sequenced from the phototrophic bacterium Rhodobacter capsulatus B10. The gene codes for a protein of 214 amino acids with a molecular mass of 23,792 Da. The deduced amino acid sequence showed significant homology with the RadC proteins of Escherichia coli, Bacillus subtilis and Haemophilus influenzae. Northern blot analysis indicated that under both chemotrophic and phototrophic growth conditions the radC gene was relatively highly expressed and was induced about five-fold after UV-irradiation. Primer extension analysis revealed that transcription was initiated from the same position before and after UV treatment. Mutants (radC negative) have a low survival rate and a slower growth rate than the wild type.