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. This year we developed an assay that detects misincorporation errors during transcription. That has been a goal for over 50 years, but such transcription errors are so rare and the consequences generally so transient that they have not been detectable. Our previous approach involved capturing the errors as DNA events by using retrotransposons. This year we created an assay that uses a defect in the active site codon for the site specific recombinase, cre. Because cre recombinase acts as a tetramer, single translation errors can not suppress the defect. This approach reduced the noise from translation errors below the frequency of transcription errors and allowed us to observe transcription errors as activation of the cre recombinase which in is detected as a permanent consequence on a selectable recombination reporter. Using this approach we have identified several RNA polymerase mutations that cause increased base substitution errors in transcription. Our goal is to expand this approach to help define the features of RNA polymerase that control its fidelity and to use error prone mutants to allow the analysis of the consequences of errors in transcription. 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. This year we developed an assay for a template switching error during retrotransposition. We will use that to identify HIV-RT alleles that have altered error rates of that kind.
