Transfer RNA molecules form an essential component of the protein synthesis system by supplying the correct amino acids to the growing polypeptide chain in a codon-specific manner. In this functional role, the level of aminoacyl-tRNAs in the cell is important in governing the rate of protein synthesis; a particularly dramatic illustration of this fact is the physiologic response of stringent control in E. coli. The cellular consequences of a deficiency in the level of any one aminoacyl-tRNA is the rapid shutdown of protein synthesis; in addition, a wide-ranging set of other processes are affected. Thus, the extent of charging is an important regulatory signal to the cell as a whole, and it is reasonable to assume that mechanisms exist in E. coli to regulate and maintain the level of all aminoacyl-tRNAs. The long term goal of the proposed research is to understand the regulation of tRNA levels in E. coli as an integral part of the maintenance of aminoacyl-tRNA levels in the cell. To this end, a hypothesis of coordinate autoregulation of amino acid biosynthetic operons, tRNA operons and aminoacyl-tRNA synthetase operons is formulated. In this scheme, tRNA genes are subject to repression by their cognate synthetases in response to the level of the cognate charged tRNA. This hypothesis was tested in in vitro transcription assays of the cloned tRNA-Arg operon by adding a crude mixture of synthetases that have been made nuclease-free. Preliminary evidence suggests the existence of a repressor protein in the synthetase fraction. Furthermore, the repressor protein alone is insufficient for repression; the presence of both tRNAs and amino acids (specifically, arginine) is required. Thus, it appears that arginyl-tRNA synthetase might be a repressor of the tRNA-Arg operon, requiring arginyl-tRNA as the co-repressor. Experiments are proposed herein to clearly define this repression mechanism as to the identity of the repressor protein and the co-repressor molecule. Optimally effective concentrations of repressor, tRNAs and arginine will be determined by in vitro transcription assays. The DNA signal responsible for repressor binding will be elucidated by DNase I footprinting technique.
