A fluorescent assay for the genetic dissection of the RNA polymerase II termination machinery

RNA polymerase II is a highly processive enzyme that synthesizes mRNAs and some non-protein coding RNAs. Termination of transcription, which entails release of the transcript and disengagement of the polymerase, requires an active process. In yeast, there are at least two multi-protein complexes needed for termination of transcription, depending upon which class of RNAs are being acted upon. In general, the two classes are relatively short non-coding RNAs (e.g. snoRNAs) and relatively long mRNAs, although there are exceptions. Here, a procedure is described in which defective termination can be detected in living cells, resulting in a method that allows strains with mutations in termination factors or cis-acting sequences, to be identified and recovered. The strategy employs a reporter plasmid with a galactose inducible promoter driving transcription of green fluorescent protein which yields highly fluorescent cells. When a test terminator is inserted between the promoter and the fluorescent protein reading frame, cells fail to fluoresce. Mutant strains that have lost termination capability, so called terminator-override mutants, gain expression of the fluorescent protein and can be collected by fluorescence activated cell sorting. The strategy is robust since acquisition of fluorescence is a positive trait that has a low probability of happening adventitiously. Live mutant cells can easily be cloned from the population of positive candidates. Flow sorting is a sensitive, high-throughput detection step capable of discovering spontaneous mutations in yeast with high fidelity.