Ret1-1, a Yeast Mutant Affecting Transcription Termination by RNA Polymerase III

In eukaryotes, extended tracts of T residues are known to signal the termination of RNA polymerase III transcription. However, it is not understood how the transcription complex interacts with this signal. We have developed a selection system in yeast that uses ochre suppressors weakened by altered transcription termination signals to identify mutations in the proteins involved in termination of transcription by RNA polymerase III. Over 7600 suppression-plus yeast mutants were selected and screened, leading to the identification of one whose effect is mediated transcriptionally. The ret1-1 mutation arose in conjunction with multiple rare events, including uninduced sporulation, gene amplification, and mutation. In vitro transcription extracts from ret1-1 cells terminate less efficiently at weak transcription termination signals than those from RET1 cells, using a variety of tRNA templates. In vivo this reduced termination efficiency can lead to either an increase or a further decrease in suppressor strength, depending on the location of the altered termination signal present in the suppressor tRNA gene. Fractionation of in vitro transcription extracts and purification of RNA polymerase III has shown that the mutant effect is mediated by highly purified polymerase in a reconstituted system.