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.
