While the central role of transcription in cell and molecular biology has been recognized for over 50 years, there has been no successful approach to the study of what controls the fidelity of the process or the consequences of infidelity in transcription. We developed methods to monitor the fidelity of transcription and the functions that contribute to the accuracy of that process. One such method involves monitoring the fidelity of retrotransposition. We isolated mutations in a subunit of RNA polymerase that reduce the fidelity of retrotransposition and demonstrated that they directly affect the accuracy of transcription. These are the first eukaryotic mutations known to reduce the fidelity of transcription. We also developed a screen for RNA polymerase mutants that increase the frequency of slippage during transcription. This class of transcription error has been shown to occur in bacteria and humans, but the features that avoid such errors have not been determined. With that screen we isolated the first mutations that increase errors of this type. We are investigating the biological consequences of increased transcription error rates. We have demonstrated that the combination of an error prone RNA polymerase with a defect in editing transcription errors is a lethal combination. We used a related assay that monitors the accuracy of retrotransposition to identify mutations that alter the fidelity of the HIV-1 reverse transcriptase. Our system uses a hybrid retrotransposon that uses the HIV-1 RT to propagate a yeast retroelement. We developed an assay that detects errors made during retrotransposition and applied it as a screen of variants of the HIV-1 RT gene. This approach identified a new class of mutations that reduce the accuracy of HIV-1 RT. These mutations, when put back into the HIV virus reduce its ability to replicate. They may define a new target for retroviral inhibitors.
