Published online 26 July 2005
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The involvement of the aspartate triad of the active center in all catalytic activities of multisubunit RNA polymerase
1Public Health Research Institute 225 Warren Street, Newark, NJ 07103, USA 2Department of Molecular Biology and Biochemistry, Waksman Institute, Rutgers University Piscataway, NJ 08854, USA 3A.N. Belozersky Institute, Moscow State University Moscow, Russia 4Institute of Molecular Genetics, Russian Academy of Sciences Moscow, Russia 123182
*To whom correspondence should be addressed. Tel: +1 732 445 6095; Fax: +1 732 445 5735; Email: severik{at}waksman.rutgers.edu
Received April 2, 2005. Revised June 6, 2005. Accepted June 14, 2005.
Three conserved aspartate residues in the largest subunit of multisubunit RNA polymerases (RNAPs) coordinate two Mg2+ ions involved in the catalysis of phosphodiester bond synthesis. A structural model based on the stereochemistry of nucleotidyl transfer reaction as well as recent crystallographic data predict that these Mg2+ ions should also be involved in the reverse reaction of pyrophosphorolysis as well as in the endo- and exonucleolytic cleavage of the nascent RNA. Here, we check these predictions by constructing point substitutions of each of the three Asp residues in the ß' subunit of Escherichia coli RNAP and testing the mutant enzymes' functions. Using artificially assembled elongation complexes, we demonstrate that substitutions of any of the three aspartates dramatically reduce all known RNAP catalytic activities, supporting the model's predictions that same amino acids participate in all RNAP catalytic reactions. We demonstrate that though substitutions in the DFDGD motif decrease Mg2+ binding to free RNAP below detection limits, the apparent affinity to Mg2+ in transcription complexes formed by the mutant and wild-type RNAPs is similar, suggesting that NTP substrates and/or nucleic acids actively contribute to the retention of active center Mg2+.
Correspondence may also be addressed to Arkady Mustaev. Tel: +7 973 854 3440; Fax: +7 973 854 3441; Email: arkady{at}phri.org
The authors wish to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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