DO TRANSCRIPTION ERRORS BY RNA POL II CONTRIBUTE TO VARIATION IN HIV

当整合到人类基因组中时，HIV-1基因组被转录 通过RNA聚合酶II产生信使和病毒基因组 RNA。 我们的目标是检测RNA聚合酶II的转录 可能有助于在某些部分观察到的高变化， 艾滋病毒基因组。 为了解决这个问题，我们正在确定 包括纯化的T7 RNA聚合酶 以及存在于人类细胞提取物中的RNA聚合酶II。在此 项目的第一年，我们一直在开发实验性的 战略 我们接下来使用这种策略来检查 简单的转录系统，T7 RNA聚合酶。 在测量转录错误率方面证明是成功的。T7 RNA聚合物 总碱基替换的错误率<0.3 × 10 ~（-4）。然而，在这方面， 对于特定的变化，碱基替换错误率可能高达 10exp（-2）。 据我们所知，这些结果提供了第一次测量 在体外转录过程中， 它们也提供了直接证据 局部序列上下文对错误产生有很大影响 在转录过程中。我们目前正在确定 人细胞提取物中多亚基RNA聚合酶II的保真度。 我们构建了所需的含CMV的M13 mp2载体， 启动子上游的T7 RNA聚合物，并进行转录 细胞提取物中的反应。

While integrated into the human genome, the HIV-1 genome is transcribed by the RNA polymerase II to produce both messenger and viral genomic RNAs. Our goal is to examine whether transcription by RNA polymerase II may contribute to the high variation observed in certain portions of the HIV genome. To address this question, we are determining the fidelity of transcription by RNA polymerases, including purified T7 RNA polymerase and the RNA polymerase II present in human cell extracts. During this first year of the project, we have been developing the experimental strategy. We have next used this strategy to examine the fidelity of a simple transcription system, T7 RNA polymerase.The applied strategy proved successful in measuring transcription error rates. The T7 RNA pol error rate for overall base substitutions is <0.3 x 10exp(-4). However, for specific changes the base substitution error rate maybe as high as 10exp(-2). To our knowledge these results provide the first measurements of error rates during in vitro transcription in the presence of all four nucleotides on a natural template they also provide a direct evidence that local sequence contexts have a strong effect on error production during transcription. We are currently in the process of determining the fidelity of the multi-subunit RNA polymerase II in human cell extracts. We have constructed the necessary M13mp2 vector containing the CMV promoter upstream from the T7 RNA pol and are conducting transcription reactions in cell extracts.