This work involves an investigation of the genetic control and enzymatic mechanisms involved in tRNA biosynthesis in Escherichia coli. We will utilize recombinant DNA methodology to elucidate the gene structure of three enzymes involved in tRNA biosynthesis: RNase P, tRNA nucleotidyltransferase and the RNase defined by the BN mutation of E. coli. (Seidman, et al., Cell 5, 389-400 (1975)). We will construct synthetic tRNA dimers using RNA ligase. These dimers will be tested as substrates for RNase P. In addition we will develop a novel method of photoaffinity labeling in an attempt to define the sites of protein-nucleic acid interaction during RNA processing reactions.