The objective of this proposal is to elucidate the mechanisms of regulation of heme-regulated eIF-2. kinase (HRI) at the molecular structural level. The specific aims are (1) structure-function relationship of the autokinase and eIF-2. activities if HRI; (2) molecular mechanisms in the regulation of HRI by heme. The methods to be employed are recombinant DNA techniques, polymerase chain reaction, synthesis of oligodeoxynucleotides , high pressure liquid chromatography, amino acid microsequencing. In vitro transcription and translation, protein kinase assays, gel electrophoresis, deoxynucleotide sequencing, expression of HRI protein in recombinant baculovirus, immunoprecipitation and Western Blot analysis. HRI is a key regulator of protein synthesis, a process which is essential for the regulation of cell growth, differentiation, antiviral response and hormonal responses. Structure-function analysis of HRI will advance our knowledge on the regulation of protein kinases which are vital in the regulation of various cellular processes.
