Mapping of T7 RNA polymerase active site with novel reagents - oligonucleotides with reactive dialdehyde groups

Oligonucleotides of a novel type containing 2'-O-beta-ribofuranosyl-cytidin e were synthesized and further oxidized to yield T7 consensus promoters wit h dialdehyde groups, Both types of oligonucleotides were tested as template s, inhibitors, and affinity reagents for T7 RNA polymerase and its mutants. All oligonucleotides tested retained high affinity towards the enzyme, Wil d-type T7 RNA polymerase and most of the mutants did not react irreversibly with oxidized oligonucleotides. Affinity labeling was observed only with t he promoter-containing dialdehyde group in position (+2) of the coding chai n and one of the mutants tested, namely Y639K. These results allowed us to propose the close proximity of residue 639 and the initiation region of the promoter within initiation complex. We suggest the oligonucleotides so mod ified may be of general value for the study of protein-nucleic acid interac tions, (C) 1999 Federation of European Biochemical Societies.