Two components of Saccharomyces cerevisiae transcription factor IIIB (TFIIIB) are stereospecifically located upstream of a tRNA gene and interact with the second-largest subunit of TFIIIC

Abstract

A novel photocrosslinking method has been used to identify the components of transcription factor IIIB (TFIIIB) and TFIIIC that associate with DNA upstream of the Saccharomyces cerevisiae SUP4 tRNATyr gene and to map these components to specific positions in DNA. When TFIIIC binds to the tRNA gene, only its second-largest subunit (135 kDa) is accessible for reaction with a photoactive nucleotide, 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUMP, inserted into DNA upstream of the transcriptional start. Formation of TFIII(C + B)-tRNA gene complexes specifically brings two additional polypeptides (90 and 70 kDa) within reach of upstream photoprobes. A collection of 13 probes has been used to map the locations of these three proteins along a 45-bp segment of DNA upstream of the transcriptional start site. Evidence is presented that the 90- and 70-kDa polypeptides are separate and distinct components of yeast TFIIIB, that they are accessible to crosslinking on opposite sides of the DNA helix in a 6-bp segment centered 35 bp upstream of the tRNATyr gene transcriptional start, and that they interact with the second-largest subunit of TFIIIC.