In Escherichia coli, as well as other prokaryotic and eukaryotic organisms, adaptation to a shift up in temperature requires the induction of the well-characterized heat shock response whose members include protein chaperones and peptidases. Although the best characterized change that occurs at low temperature is the decrease in the saturation of fatty acids in the membrane phospholipids, the induction of the cold shock response indicates that other physiological changes are required for adaptation. However, unlike the heat shock response, the adaptive function of the cold shock response is not understood. The long-term goal is to understand the physiological basis of the induction of the cold shock response for growth at low temperature by revealing unique requirements for growth and gene expression at low temperatures. This proposal will also increase our understanding of the role of DEAD-box proteins in cellular physiology. Members of the DEADbox family of ATP-dependent helicases are found in various living cells from bacteria to man and are characterized by 8 conserved motifs. These enzymes are involved in various cellular processes such as RNA splicing, ribosome biogenesis, translational initiation, mRNA degradation, and cell division. A DEAD-box protein of E. coli, CsdA is cold shock inducible, and has RNA helix destabilizing activity. The goal of this proposal is to characterize the function of CsdA in bacterial physiology at low temperature. CsdA is an important enzyme in cellular physiology at low temperatures. CsdA associates with the ribosome and is required for optimal growth and gene expression at low temperatures. The first objective is to assess the requirement of the motifs, as well as additional residues for the function of CsdA. Various csdA alleles will be generated by mutagenesis and tested for the ability to increase growth and gene expression at low temperatures. The second objective is to characterize the mutant proteins in vitro. The mutant proteins will be tested for various activities such as RNAunwinding/binding, ATP binding/hydrolysis. The effect of the protein on in vitro transcription and translation and on the association with other protein factors will be analyzed.
