Nuclear magnetic resonance studies on the role of intrinsic metals in Escherichia coli RNA polymerase: effect of DNA template on the nucleotide-enzyme interaction

Abstract

Nuclear magnetic resonance studies were performed to investigate the effect of DNA template on the interaction of initiating nucleotide ATP with Escherichia coli RNA polymerase (RPase) in which one of the two intrinsic Zn ions was substituted with a Co(II) (Co-Zn RPase) or Mn(II) (Mn-Zn RPase) ion. This intrinsic metal ion is located at the initiation site in the β subunit of RPase. The paramagnetic effects of Co-Zn and Mn-Zn RPases on the relaxation rates of ¹H- and ³¹P-nuclei of ATP were used to determine the distances from the intiation sites in the presence of DNA. The distances from the metal to H₂, H_a, H₁, α-P, β-P, and γ-P atoms were estimated to be 6.7±0.9, 4.1±0.6, 6.1±1.2. These distances were compared with those measeured in the absence of DNA (Chatterji, D., and Wu, F. Y.-H.(1982) Biochemistry 21, 4657). In both the presence and absence of DNA, the close proximity between the intrinsic metal and the H₈ atom strongly indicates that the metal is coordinated directly to the base moiety of ATP. Such a coordination may provide a structural basis for the selection of a purine nucleotide during the initiation process. The presence of DNA causes the H₂atom to move away (> 2 Å) from the intrinsic metal, whereas all three phosphorus atoms shift closer (>3 Å) toward the metal. The possible mechanistic implications of the conformational alteration of ATP at the initiation site induced by the DNA template is discussed.