MAPPING OF CATALYTIC RESIDUES IN THE RNA-POLYMERASE ACTIVE-CENTER

When the Mg2+ ion in the catalytic center of Escherichia coli RNA poly merase (RNAP) is replaced with Fe2+, hydroxyl radicals are generated. In the promoter complex, such radicals cleave template DNA near the tr anscription start site, whereas the beta' subunit is cleaved at a cons erved motif NADFDGD (Asn-Ala-Asp-Phe-Asp-Gly-Asp). Substitution of the three aspartate residues with alanine creates a dominant lethal mutat ion. The mutant RNAP is catalytically inactive but can bind promoters and form an open complex, The mutant fails to support Fe2+-induced cle avage of DNA or protein. Thus, the NADFDGD motif is involved in chelat ion of the active center Mg2+.