SELECTIVITY OF ESCHERICHIA-COLI RNA-POLYMERASE FOR TEMPLATE CONFORMATION

Escherichia coli RNA polymerase (RNAP) exhibits a strong selectivity for the secondary structure of its template DNA, as shown by the influence both of the DNA conformation on the transcription cycle and of the enzyme on the DNA conformation itself Binding, chain initiation and elongation characteristics of RNAP, and DNA conformational characteristics were examined by use of the alternating copolymer poly(dGdm5C).poly(dGdm5C) as template. Transcription is impeded when the DNA is in the Z conformation as compared with the B; the initial conformation is determined by the concentration of the conformational effectors Mg2+ and [Co(NH3)6]3+. RNAP binds to both Z and B conformers; the total binding is moderately greater when the template is in the B conformation than when it is strongly stabilized in the Z, by [Co-(NH3)6]3+ concentrations much higher than those required for B-Z transition. However, the Z conformer is much more easily displaced competitively from the bulk of its complexes with RNAP than is the B, indicating a specific binding preference for the B conformer. When the template is in the B conformation, or is moderately stabilized in the Z by Mg2+ concentrations such that the polynucleotide is just fully converted from B to Z, elongation is predicted well by chain initiation, indicating that on the Z conformer RNAP is effectively inhibited at the chain initiation or at an earlier stage. The average chain growth rates for polymeric product synthesized on B and on moderately stabilized Z are similar, even though overall RNA synthesis is considerably lowered on the Z form, again indicating that the limiting events precede elongation. When the Z conformer is strongly stabilized, chain initiation and elongation are further inhibited. Elongation is still roughly correlated with chain initiation, but some additional inhibition of elongation takes place independently. Circular dichroism analysis shows that RNAP-DNA binding affects the B-Z conformational equilibrium, leading to reformation of the B conformer from Z and interference with conversion of B to Z, under conditions that would otherwise favor the Z conformer. Thus, there is an RNAP concentration dependent shift of the B-Z transition to higher concentrations of Z-inducing cation, and there is an RNAP concentration dependent decrease in the rate of B to Z conversion. These effects were observed for poly-(dGdm5C).poly(dGdm5C), with Z stabilized by [Co(NH3)6]3+ or Mg2+. (They were observed as well for the unmethylated copolymer poly(dGdC).poly(dGdC), with Z stabilized by [Co(NH3)6]3+.) Perturbation of the Z conformer was detectable by circular dichroism at an RNAP:polynucleotide ratio down to a practical limit of approximately 1 RNAP:500 bp. Interference with B to Z conversion was readily detected at 1 RNAP:500 bp. Both of these phenomena appear to be fairly specific for the holoenzyme (alpha-2-beta-beta'sigma) form of RNAP, with the core enzyme (alpha-2-beta-beta') giving much smaller effects.