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.
