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

工作总结：这个项目的目标有两个：检查RNA聚合酶II的转录是否可能导致在HIV基因组的某些部分观察到的高变异，以及调查转录不忠诚是否可能影响细胞基因组的稳定性。为了解决这些问题，我们开发了一种体外系统，可以定量评估在天然模板上合成过程中人类细胞提取物中RNA聚合酶的保真度，包括纯化的T7 RNA聚合酶(RNAP)和RNA PolII，在所有四个核苷酸的存在下。最近，我们利用这个系统来研究非转录链在T7RNA Poll转录保真度中的作用。初步结果表明，非模板链中的未配对碱基影响转录的移码频率。在非转录链中含有4个核苷酸环的DNA的转录导致2碱基添加的频率相对于同源双链DNA上的添加频率增加了16倍。这一观察表明，RNA聚合酶与非转录链的相互作用影响转录的保真度。事实上，RNA聚合酶与非转录链的相互作用影响转录的延伸，这一事实在文献中得到了很好的证明。然而，据我们所知，这是第一个表明RNA聚合酶与非转录链之间的相互作用可能影响转录保真度的结果。上述结果也提出了一个问题，即对不受转录偶联修复影响的非转录单链的巨大损伤是否会改变转录的保真度。我们正在设计实验来检验这种可能性。在本财年期间，我们还致力于提高人类细胞核提取物的转录效率。这些努力都没有成功。因此，由于缺乏足够数量的信使核糖核酸用于进一步分析，我们决定终止从人类细胞中提取细胞核的工作。我们计划用纯化的大肠杆菌RNA聚合酶进行测量，它与RNA PolII具有广泛的同源性。这将是与北卡罗来纳大学的Dorothy Erie博士合作的努力。

Summary of Work: The objective of this project is two fold: to examine whether transcription by RNA polymerase II may contribute to the high variation observed in certain portions of the HIV genome and to investigate if transcriptional infidelity may affect the stability of the cellular genome. To address these questions we have developed an in vitro system that allows quantitative evaluation of the fidelity of RNA polymerases including purified T7 RNA polymerase(RNAP) and RNA pol II in human cell extracts during synthesis on a natural template in the presence of all four nucleotides. Recently we have employed this system to examine the role of the non-tanscribed strand on the fidelity of transcription by T7 RNA pol. The initial results indicate that unpaired bases in the non-template strand affect the frameshift frequency of transcription. Transcription of DNA containing a 4-nucleotide loop in the non-transcribed strand resulted in a 16-fold increase in the frequency of 2-base additions relative to the frequency of additions on homoduplex DNA. This observation suggests that interactions of the RNA polymerase with the non-transcribed strand affect the fidelity of transcription. The fact that interactions of the RNA polymerase with the non-transcribed strand influence elongation of transcription is well documented in the literature. However, to our knowledge this is the first result suggesting that interactions between RNA polymerase and the non-transcribed strand may affect fidelity of transcription. The above result also raises the question whether bulky damage on the non-transcribed strand, which is not subject to transcription-coupled repair, may modify the fidelity of transcription. We are in the process of designing experiments to examen this possibility. During this FY we have also worked on improving the transcriptional efficiency of nuclear extracts from human cells. These efforts were unsuccessful. Thus, due to the lack of sufficient quantity of mRNA for further analysis we decided to terminate the work with nuclear extracts from human cells. We plan to perform measurement with purified E. coli RNA polymerase which has extensive homology with RNA pol II. This will be a collaborative effort with Dr. Dorothy Erie from UNC.