Eukaryotic RNA polymerase III is responsible for the transcription of many small RNA molecules, including all cytoplasmic tRNAs. Primary recognition specificity of tRNA genes by the PolIII transcription complex resides in two or more proteins which are present in chromatin but can be separated from RNA polymerase III in whole cell extracts. Formation of a pre-initiation complex between a tRNA gene and these proteins is prevented by mutations which alter sequences within the Dihydro U and T Psi arms of tRNA coding sequence. The goal of this research project is to dissect apart and identify the various DNA-protein interactions which occur during the initiation and termination of transcription of eukaryotic tRNA genes. Ochre-suppressor tRNA genes in yeast provide unique opportunities to isolate and study both mutations in tRNA genes that affect transcription and mutations in genes for protein components that interact with the genes during transcription. It is envisioned that the genetic analysis of such mutations affecting expression of the yeast SUP4 tRNATyr gene, together with biochemical studies of the proteins affected by these mutations, will provide new insights into the transcription process. As this approach is developed, it should also be applicable to molecular genetic analysis of the function of RNA Polymerase II, the enzyme which makes mRNA in yeast and other eukaryotes.
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