We have shown that during normal transcription E. coli RNA polymerase is capable of removing the nucleotide which it has just added to a growing RNA chain and that this removal depends on the presence of Small concentrations of pyrophosphate (PPi). After the removal the enzyme can continue synthesis. The removal reaction is probably simply the reversal of the incorporation reaction, and we have observed the generation of free triphosphate as a result. We have written a theory which proposes that this reversibility may increase the discrimination free energy between correct and incorrect nucleotides and therefore may increase the fidelity of transcription. The formation of a covalent phosphodiester bond allows discrimination on the basis of helical structure as well as base-pairing. We propose that the important discrimination step is the translocation of the enzyme from one site on the DNA template to the next, and that reversible incorporation is necessary in order to take full advantage of the maximum discrimination free energy. We are requesting funds to test this hypothesis.
| Kahn, J D; Hearst, J E (1989) Reversibility of nucleotide incorporation by Escherichia coli RNA polymerase, and its effect on fidelity. J Mol Biol 205:291-314 |
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