Mapping of catalytic residues in the RNA polymerase active center.
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+.
Zaychikov
E
E
Limnological Institute, Russian Academy of Sciences, Irkutsk, Russia.
Martin
E
E
Denissova
L
L
Kozlov
M
M
Markovtsov
V
V
Kashlev
M
M
Heumann
H
H
Nikiforov
V
V
Goldfarb
A
A
Mustaev
A
A
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000378:metabolism; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D005296:Ferrous Compounds / Q000378:metabolism; D017665:Hydroxyl Radical; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011401:Promoter Regions, Genetic
D005296:Ferrous Compounds; D001224:Aspartic Acid; D017665:Hydroxyl Radical; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D008274:Magnesium; D004229:Dithiothreitol