The E.coli RNA degradosome analysis of molecular chaperones and enolase

Normal mRNA turnover is essential for genetic regulation within cells. The E. coli RNA degradosome, a large multi-component protein complex which originates through specific protein interactions, has been referred to as the “RNA decay machine” and is responsible for mRNA turnover. The degradosome functions to process RNA and its key components have been identified. The scaffold protein is RNase E and it tethers the degradosome to the cytoplasmic membrane. Polynucleotide phosphorylase (PNPase), ATP-dependent RNA helicase (RhlB helicase) and the glycolytic enzyme enolase associate with RNase E to form the degradosome. Polyphosphate kinase associates with the degradosome in substoichiometric amounts, as do the molecular chaperones DnaK and GroEL. The role of DnaK as well as that of enolase in the RNA degradosome is unknown. Very limited research has been conducted on the components of the RNA degradosome under conditions of stress. The aim of this study was to understand the role played by enolase in the assembly of the degradosome under conditions of stress, as well as investigating the protein levels of molecular chaperones under these conditions. The RNA degradosome was successfully purified through its scaffold protein using nickel-affinity chromatography. In vivo studies were performed to investigate the protein levels of DnaK and GroEL present in the degradosome under conditions of heat stress, and whether GroEL could functionally replace DnaK in the degradosome. To investigate the recruitment of enolase to the degradosome under heat stress, a subcellular fractionation was performed to determine the localization of enolase upon heat shock in vivo. The elevated temperature resulted in an increased concentration of enolase in the membrane fraction. To determine whether there is an interaction between enolase and DnaK, enolase activity assays were conducted in vitro. The effect of DnaK on enolase activity was measured upon quantifying DnaK and adding it to the enolase assays. For the first time it was observed that the activity of enolase increased with the addition of substoichiometric amounts of DnaK. This indicates that DnaK may be interacting with the RNA degradosome via enolase