The rnc operon of Escherichia coli encodes two interesting and important functions: ribonuclease III and Era. The membrane-associated Era protein is a member of an important class of regulatory proteins whose activities are modulated by the binding and hydrolysis of guanine nucleotides. These G-proteins are present in all living organisms and participate in many different cellular functions, all of which involve signal transduction. Era shares sequence similarity with yeast RAS (hence, E. coli Ras analogue), a member of the """"""""small"""""""" G-protein subfamily that includes the mammalian p2lras protooncogene. Because of Era's membership in this subfamily and its essential role in E. coli physiology, it too is receiving considerable attention. The extensive work on p2lras, E. coli translation elongation factor Tu and eukaryotic heterotrimeric G-proteins has produced a fairly detailed picture of their structure and function. The conserved sequences for GTP binding and hydrolysis and the striking similarity between p2lras and EF-Tu crystal structures indicate that these structure-function relationships are highly conserved. RNaseIII is one of the best characterized endoribonucleases and the primary if not sole double-stranded RNA specific endonuclease in E. coli. Essentially identical or very similar activities are found in a wide variety of cells, and RNaseIII is a member of the newly discovered and important family of double-stranded RNA binding proteins, which includes the HIV TAR-binding protein and the human double-stranded RNA-dependent kinase. RNaseIII's involvement in stable RNA processing and control of gene expression at the RNA level is well established. Indeed, RNaseIII regulates rnc operon expression by a complex and yet to be defined posttranscriptional mechanism. Recent evidence suggests that RNaseIII may perform important roles in essential cell functions, most likely in concert with Era. One long-term goal of this proposal is to define the structure and function of RNaseIII, and to elucidate its role in cell viability. Work proposed here will include an analysis of the mechanism of rnc operon regulation. the isolation and detailed characterization of a variety of mutations affecting different RNaseIII functions, and the isolation and study of RNaseIII homologues from related organisms. These studies should clarify the putative interplay between RNaseIII and Era and provide important insights into the nature of double-stranded RNA binding and cleavage. A second long-term goal of this proposal is to develop a better understanding of the mechanisms of G-protein function. Work proposed here will include a systematic genetic analysis of Era to determine structure- function relationships and to identify the components of the Era signal transduction pathway. These studies will be aided by ongoing collaborative efforts to identify Era homologues from other organisms and to determine the Era crystal structure. Results from this work should provide significant insights into the function of oncogenic p2lras and other G- proteins.
