Reversibility of nucleotide incorporation by Escherichia coli RNA polymerase, and its effect on fidelity.

During transcription, Escherichia coli RNA polymerase is capable of removing the nucleotide that it has just added to a growing RNA chain, and this removal depends on the presence of small concentrations of pyrophosphate. Chemically, the removal reaction is simply the reversal of the incorporation reaction, and we have observed the generation of free triphosphate as a result. After the removal the enzyme can continue synthesis. To test whether this reaction can provide an error correction mechanism, misincorporation rates were measured at a single position in an RNA transcript by withholding the correct nucleotide for that position, measuring the amount of readthrough transcript, and analyzing the readthrough transcripts with nearest-neighbor analysis and enzymatic RNA sequencing. The removal of pyrophosphate increases the rate of misincorporation. We present a theory that explains how reversible incorporation can increase the available 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.