The relative abundance of different tRNAs is statically regulated by the sequence and spacing of internal control regions (ICR), both 5' and 3' - flanking sequences, and the copy number of individual tRNA genes. A protein factor, TFIIIC, nucleates formation of transcription complex. Variations from canonical sequences and spacing in the ICR are known to affect this interaction which may determine which genes are expressed. We have participated in development of a photosensitive cross-linking reagent, 5-N3- deoxyuridine-5'-triphosphate (AdUTP), which can be enzymatically incorporated into DNA in place of thymidine. The photoactive DNA is capable of sequence-specific interaction with TFIIIC and can be used to identify the subunit of this protein which contacts the B-block of the ICR. We propose to study the interactions of this protein with tRNA genes to identify which T residues are in close association with TRIIIC, whether A & B blocks contact the same or different subunits, whether other T residues in the coding or flanking sequences can be cross-linked to the factor, and what regions of the protein contact the ICR. The gene(s) for the DNA binding subunit (s) will be identified in a yeast genomic library using synthetic DNA probes based on partial amino acid sequence analysis of purified polypeptides. The nucleotide sequence of this gene (s) will be determined to deduce the amino acid sequence analysis of purified polypeptides. The nucleotide sequence of this gene (s) will be determined to deduce the amino acid sequence. Peptide mapping of TFFIIIC photocrosslinked to AdUTP substituted DNA will allow us to identify sites in the protein which are in close contact with each tRNA control region. Studies of mutations in 5'-flanking and anticodon sequences which affect tRNA gene expression will also be completed. These investigations deal with the factors that control the formation of transfer RNA's, (tRNA's), which are ribonucleic acid molecules involved in transferring amino acids to nascent peptide chains. One of these factors is a protein called TFIIIC that reacts with the genes that code for these tRNA's. The investigations will employ a base that will substitute for thymine in DNA and react with TFIIIC in the presence of light. In this way, the site on the DNA with which TFIIIC reacts can be identified.
