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J. Biol. Chem., Vol. 275, Issue 33, 25516-25522, August 18, 2000

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Dissection of Two Hallmarks of the Open Promoter Complex by Mutation in an RNA Polymerase Core Subunit^*

Sergei Nechaev, Mark Chlenov^§, and Konstantin Severinov^¶

From the  Waksman Institute and Department of Genetics, Rutgers, The State University, Piscataway, New Jersey 08854 and the ^§ Institute of Molecular Genetics, Moscow 123182, Russia

Deletion of 10 evolutionarily conserved amino acids from the  subunit of Escherichia coli RNA polymerase leads to a mutant enzyme that is unable to efficiently hold onto DNA. Open promoter complexes formed by the mutant enzyme are in rapid equilibrium with closed complexes and, unlike the wild-type complexes, are highly sensitive to the DNA competitor heparin (Martin, E., Sagitov, V., Burova, E., Nikiforov, V., and Goldfarb, A. (1992) J. Biol. Chem. 267, 20175-20180). Here we show that despite this instability, the mutant enzyme forms partially open complexes at temperatures as low as 0 °C when the wild-type complex is fully closed. Thus, the two hallmarks of the open promoter complex, the stability toward a challenge with DNA competitors and the sensitivity toward low temperature, can be uncoupled by mutation and may be independent in the wild-type complex. We use the high resolution structure of Thermus aquaticus RNA polymerase core to build a functional model of promoter complex formation that accounts for the observed defects of the E. coli RNA polymerase mutants.

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