IDENTIFICATION OF THE TEMPLATE-BINDING CLEFT OF T7 RNA-POLYMERASE AS THE SITE FOR PROMOTER-BINDING BY PHOTOCHEMICAL CROSS-LINKING WITH PSORALEN

We describe a novel method of photo-cross-linking DNA-binding proteins to DNA employing psoralen as a tether, We apply this method for the i nteraction of T7 RNA polymerase to its promoter. The crystallographic model of T7 RNA polymerase shows a cleft formed by the palm, thumb, an d fingers domains, It was proposed that template DNA binds in the clef t. Here we directly and positively identify, in solution, the cleft as the seat of template binding. We photo-cross-linked a 23 bp promoter DNA to T7 RNA polymerase. We then determined the masses of cross-linke d tryptic peptides by mass spectrometry and analyzed their amino acid composition. The cross-linked peptides were projected on the crystal s tructure of T7 RNA polymerase. The peptides nicely decorated the back, front, and side wall of the cleft. In a previous work [Sastry et al. (1993) Biochemistry 32, 5526-5538] we used site-specific psoralen fura n-side monoadducts for cross-linking DNAs to DNA-binding proteins. We cross-linked a single-stranded 12-mer oligonucleotide to T7 RNA polyme rase. We isolated and purified a DNA cross-linked tryptic peptide. We then used mass spectrometry and amino acid composition analysis to ide ntify the location of this peptide on the T7 RNA polymerase primary se quence, In the present work we have mapped this peptide on the 3-D str ucture of T7 RNA polymerase. This peptide maps in the fingers domain o f the polymerase. On the basis of a comparison of the map positions of peptides that crosslinked to either promoter DNA or single-stranded o ligo-DNA, we propose that different functional domains may be involved in binding of double-stranded promoter DNA and nonspecific single-str anded DNA, Whereas the cleft of the polymerase is the seat of double-s tranded promoter binding, the fingers domain may be used by the polyme rase to grab single-stranded DNA (or RNA) in a nonspecific manner. Alt ernatively, the single-stranded oligo binding site may be an RNA produ ct-binding site during transcription. The photochemical techniques we have developed [Sastry et al, (1993) Biochemistry 32, 5526-5538; this work] can be applied to other DNA-protein complexes to map DNA-binding domains.