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

The Fe 2؉ ion that specifically replaces Mg 2؉ in the active center of RNA polymerase generates reactive hydroxyl radicals that cause highly localized cleavage of polypeptide chains. Mapping of the cleavage sites revealed the overall architecture of the active center. Nine distinct sites, five in the ␤ subunit and four in the ␤ subunit of Escherichia coli RNA polymerase, all at or near highly conserved sequence motifs, are brought together in the enzyme's ternary structure within the distance of Ϸ1 nm from the active center Me 2؉ . These sites are located in at least six different domains of the subunits, ref lecting modular organization of the active center.During the past decade, x-ray crystallographic analysis of small, single-subunit polymerases, such as DNA polymerase I, HIV reverse transcriptase, and RNA polymerase of bacteriophage T7 revealed a common principal structure whereby a single protein domain contains a characteristic cleft where the enzymatic reaction of phosphodiester bond formation is believed to occur (1). A similar cleft has been observed in the low-resolution contours of large multisubunit cellular RNA polymerases (RNAPs) (2-4), leading to speculation that their active center resembles that of the smaller enzymes. The excess protein in large polymerases, which amounts to hundreds of thousands of dalton, is explained by complex regulatory requirements of the cell.But how are the larger polymerases built? Two principal types of organization are possible. First, a single catalytic domain structurally analogous to a single-subunit polymerase may be surrounded by regulatory components. Alternatively, different structural domains may participate in the active center of the large enzymes, forming the cleft that resembles that of the single-domain polymerases only in shape but not in structural design. Lack of sequence conservation between large and small polymerase families argues for the latter possibility. Here we present direct structural evidence to the same effect using the recently described technique of Fe 2ϩ -mediated local protein cleavage (5). MATERIALS AND METHODSHistidine-tagged RNA polymerase was derivatized at Met-932 of the ␤Ј subunit (V.M., E. Lukhtanov, A.G., and A.M., unpublished data) and Lys-1242 (M.K., K. Severinov, A.G., and A.M., unpublished data) of the ␤ subunit as described (5, 6), except that NaBH 4 was added after [␣-32 P]UTP. Fe 2ϩ induced autocleavage, and degradation of RNAP polypeptides at Met and Cys were performed as in (5). Products were analyzed by SDS͞PAGE and gel slabs were autoradiographed as described (5). Gel concentrations are indicated in the figure legends. RESULTS AND DISCUSSIONFe 2؉ -Mediated Cleavage of RNAP. The Fe 2ϩ ion in appropriate ligation under aerobic conditions generates hydroxyl radicals. These activated oxygen species are extremely reactive and cause degradation of biopolymers with a diffusion limited rate, within the range of Ϸ1 nm. The process is known as Fenton reaction (7) and is used for footprinting of nucleic acid-protein c...