Mapping of Catalytic Residues in the RNA Polymerase Active Center

Science  05 Jul 1996:
Vol. 273, Issue 5271, pp. 107-109
DOI: 10.1126/science.273.5271.107


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 β′ 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 NADFDGD motif is involved in chelation of the active center Mg2+.

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