Minor groove DNA-protein contacts upstream of a tRNA gene detected with a synthetic DNA binding ligand

Transcription factor IIIB (TFIIIB) is composed of the TATA box binding prot ein (TBP) and class III gene-specific TBP-associated factors (TAFs). TFIIIB is brought to a site centered approximately 35 bp upstream from the transc ription start site of tRNA genes via protein-protein interactions with the intragenic promoter-recognition factor TFIIIC. Since TBP interacts with TAT A elements through the minor groove of DNA, we asked whether TFIIIB interac ts with DNA in the minor groove. Polyamides containing pyrrole (Py) and imi dazole (Im) amino acids are synthetic DNA ligands that bind to predetermine d sequences in the minor groove of double helical DNA. These small molecule s have been shown to interfere with protein-DNA interactions in the minor g roove. A series of DNA constructs was generated in which the binding site f or a Py-Im polyamide was placed at various distances upstream from a tRNA g ene transcription start site. We find that a match polyamide will effective ly inhibit tRNA gene transcription when its binding site is located within 33 bp of the transcription start site of the Xenopus Tyro tRNA gene. Moreov er, in the presence of polyamide, RNA polymerase III is redirected to a new transcription initiation site located approximately one DNA helical turn d ownstream from the native start site. Our results suggest that a subunit of TFIIIB, possibly TBP, makes an essential minor groove DNA contact centered approximately 30 bp upstream from the tRNA gene. (C) 1999 Academic Press.