Kahn J D, Hearst J E
Department of Chemistry, University of California, Berkeley 94720.
J Mol Biol. 1989 Jan 20;205(2):291-314. doi: 10.1016/0022-2836(89)90342-2.
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.
在转录过程中，大肠杆菌RNA聚合酶能够去除它刚刚添加到正在生长的RNA链上的核苷酸，而这种去除取决于低浓度焦磷酸的存在。从化学角度来看，去除反应仅仅是掺入反应的逆反应，并且我们已经观察到会产生游离三磷酸。去除之后，酶可以继续进行合成。为了测试这种反应是否能提供一种纠错机制，通过在RNA转录本的单个位置扣留正确的核苷酸、测量通读转录本的量，并使用邻位分析和酶促RNA测序来分析通读转录本，从而测定了该位置的错误掺入率。焦磷酸的去除会增加错误掺入率。我们提出了一种理论，该理论解释了可逆掺入如何能够增加正确与错误核苷酸之间可用的辨别自由能，因此可能会提高转录的保真度。共价磷酸二酯键的形成允许基于螺旋结构以及碱基配对进行辨别。我们认为重要的辨别步骤是酶从DNA模板上的一个位点转移到下一个位点，并且可逆掺入是充分利用最大辨别自由能所必需的。
