Probing contacts of phosphate groups of oligonucleotides from the non-template strand of lac UV5 promoter with E-coli RNA polymerase using regioselective cross-linking

Contacts of phosphate groups at positions -12, -15, and -18 in relation to the transcription initiation site in the non-template strand of lac UV5 pro moter with lysines or histidines off. coli RNA polymerase in the open compl ex model were studied. A number of synthetic oligonucleotides from the -10- area of the non-template strand containing activated 5'-terminal phosphate group were cross-linked with holo- or core-enzyme of RNA polymerase. 5'-N-H ydroxybenzotriazole phosphodiesters of oligonucleotides were used as phosph ate activated derivatives. They are capable of phosphorylating amino groups of lysines and histidines in the enzyme molecule that are brought into pro ximity with activated phosphate in the complex, resulting in the formation of a covalent bond between the oligonucleotide and the protein. The analysi s of the products of cross-linking allowed the protein subunit and the amin o acid residue taking part in the formation of the covalent bond for each o ligonucleotide to be identified. It was found that all oligonucleotides fro m the non-template strand of promoter in the complex with the hole-enzyme a re bound with the sigma(70)-subunit. When analyzing the products of partial cleavage of the complexes cross-linked at cysteines and methionines using SDS-PAGE, it was shown that phosphate at position -12 made contacts with Hi s180 or His242 of the sigma(70)-subunit, the reactive amino acid residue be ing located between the first and second conservative regions. Phosphate at position -15 is located near lysines from two different areas-between Met4 13 and Met456 (regions 2.3 and 2.4) and between Met470 and Met507 (region 3 .1). Phosphate at position -18 makes preferential contacts with a lysine si tuated between Met470 and Met507 (region 3.1). Based on the analysis of con tacts of phosphate groups and the structure of the isolated sigma(79)-subun it established previously, a scheme of the mutual arrangement of the oligon ucleotide and the sigma(70)-subunit possessed by the hole-enzyme has been p roposed.