DafA Cycles Between the DnaK Chaperone System and Translational Machinery

Abstract

DafA is encoded by the dnaK operon of Thermus thermophilus and mediates the formation of a highly stable complex between the chaperone DnaK and its co-chaperone DnaJ under normal growth conditions. DafA Tth contains 87 amino acid residues and is the only member of the DnaK Tth chaperone system for which no corresponding protein has yet been identified in other organisms and whose particular function has remained elusive. Here, we show directly that the DnaK Tth–DnaJ Tth–DafA Tth complex cannot represent the active chaperone species since DafA Tth inhibits renaturation of firefly luciferase by suppressing substrate association. Since DafA Tth must be released before the substrate proteins can bind we hypothesized that free DafA Tth might have regulatory functions connected to the heat shock response. Here, we present evidence that supports this hypothesis. We identified the 70 S ribosome as binding target of free DafA Tth. Our results show that the association of DafA Tth and 70 S ribosomes does not require the participation of DnaK Tth or DnaJ Tth. On the contrary, the assembly of DnaK Tth–DnaJ Tth–DafA Tth and ribosome–DafA Tth complexes seems to be competitive. These findings strongly suggest the involvement of DafA Tth in regulatory processes occurring at a translational level, which could represent a new mechanism of heat shock response as an adaptation to elevated temperature.