Dissection of two hallmarks of the open promoter complex by mutation in an RNA polymerase core subunit

Deletion of 10 evolutionarily conserved amino acids from the beta 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 degrees 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.     

Exploitation of a chemical nuclease to investigate the location and orientation of the Escherichia coli RNA polymerase alpha subunit C-terminal domains at simple promoters that are activated by cyclic AMP receptor protein

The C-terminal domain of the alpha subunit (alphaCTD) of bacterial RNA polymerase plays an important role in promoter recognition. It is known that alphaCTD binds to the DNA minor groove at different locations at different promoters via a surface-exposed determinant, the 265 determinant. Here we describe experiments that permit us to determine the location and orientation of binding of alphaCTD at any promoter. In these experiments, a DNA cleavage reagent is attached to specific locations on opposite faces of the RNA polymerase alpha subunit. After incorporation of the tagged alpha subunits into holo-RNA polymerase, patterns of DNA cleavage due to the reagent are determined in open complexes. The locations of DNA cleavage due to the reagent attached at different positions allow the position and orientation of alphaCTD to be deduced. Here we present data from experiments with simple Escherichia coli promoters that are activated by the cyclic AMP receptor protein.     

Interaction of the alpha-subunit of Escherichia coli RNA polymerase with DNA: rigid body nature of the protein-DNA contact

The alpha-subunit of Escherichia coli RNA polymerase plays an important role in the activity of many promoters by providing a direct protein-DNA contact with a specific sequence (UP element) located upstream of the core promoter sequence. To obtain insight into the nature of thermodynamic forces involved in the formation of this protein-DNA contact, the binding of the alpha-subunit of E. coli RNA polymerase to a fluorochrome-labeled DNA fragment containing the rrnB P1 promoter UP element sequence was quantitatively studied using fluorescence polarization. The alpha dimer and DNA formed a 1:1 complex in solution. Complex formation at 25 degrees C was enthalpy-driven, the binding was accompanied by a net release of 1-2 ions, and no significant specific ion effects were observed. The van't Hoff plot of temperature dependence of binding was linear suggesting that the heat capacity change (Deltac(p)) was close to zero. Protein footprinting with hydroxyradicals showed that the protein did not change its conformation upon protein-DNA contact formation. No conformational changes in the DNA molecule were detected by CD spectroscopy upon protein-DNA complex formation. The thermodynamic characteristics of the binding together with the lack of significant conformational changes in the protein and in the DNA suggested that the alpha-subunit formed a rigid body-like contact with the DNA in which a tight complementary recognition interface between alpha-subunit and DNA was not formed.     

Open complex formation by Escherichia coli RNA polymerase: the mechanism of polymerase-induced strand separation of double helical DNA

Escherichia coli RNA polymerase is able to site-specifically melt 12 bp of promoter DNA at temperatures far below those normally associated with DNA melting. Here we consider several models to explain how RNA polymerase destabilizes duplex DNA. One popular model proposes that upon binding to the promoter, RNA polymerase untwists the spacer DNA between the –10 and –35 regions, which results in a destabilization of the –10 region at a TA base step where melting initiates. Promoter untwisting may result, in part, from extensive wrapping of the DNA around RNA polymerase. Formation of the strand-separated open complex appears to be facilitated by specific protein-DNA interactions which occur predominantly on the non-template strand. Recent evidence suggests that these include important contacts with Sigma factor region 2.3, which we propose binds the displaced single strand of DNA.