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Science 5 July 1996:
Vol. 273. no. 5271, pp. 107 - 109
DOI: 10.1126/science.273.5271.107
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Reports

Mapping of Catalytic Residues in the RNA Polymerase Active Center

Evgeny Zaychikov, Emil Martin, Ludmila Denissova, Maxim Kozlov, Vadim Markovtsov, Mikhail Kashlev, Hermann Heumann, Vadim Nikiforov, Alex Goldfarb, Arkady Mustaev *

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

E. Zaychikov and L. Denissova, Limnological Institute, Russian Academy of Sciences, Irkutsk 664033, Russia.
E. Martin, M. Kozlov, V. Markovtsov, M. Kashlev, V. Nikiforov, A. Goldfarb, A. Mustaev, Public Health Research Institute, 455 First Avenue, New York, NY 10016, USA.
H. Heumann, Max-Planck-Institut fur Biochemie, 8033 Martinsreid bei Munchen D82152, Germany.
* To whom correspondence should be addressed.


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