Use of an in vivo reporter assay to test for transcriptional and translational fidelity in yeast.
Eukaryotic RNA polymerase II and Escherichia coli RNA polymerase possess an intrinsic ribonuclease activity that is stimulated by the polymerase-binding proteins SII and GreB, respectively. This factor-activated hydrolysis of nascent RNA has been postulated to be involved in transcription elongation as well as removal of incorrect bases misincorporated into RNA. Little is known about the frequency of misincorporation by RNA polymerases in vivo or about the mechanisms involved in improving RNA polymerase accuracy. Here we have developed a luciferase reporter system in an effort to assay for base misincorporation in living Saccharomyces cerevisiae. The assay employs a luciferase open reading frame that contains a premature stop codon. The inactive truncated enzyme would become active if misincorporation by RNA polymerase II took place at the stop triplet. Yeast lacking SII did not display a significant change in reporter activity when compared with wild-type cells. We estimate that under our assay conditions, mRNAs with a misincorporation at the test site could not exceed 1 transcript per 500 cells. The reporter assay was very effective in detecting the previously described process of nonsense suppression (translational read-through) by ribosomes, making it difficult to determine an absolute level of basal (SII-independent) misincorporation by RNA polymerase II. Although these data cannot exclude the possibility that SII is involved in proofreading, they make it unlikely that such a contribution is physiologically significant, especially relative to the high frequency of translational errors.
Shaw
Randal J
RJ
Department of Biochemistry and Graduate Program in Genetics and Molecular Biology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Bonawitz
Nicholas D
ND
Reines
Daniel
D
D004926:Escherichia coli / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D017930:Genes, Reporter; D007700:Kinetics; D008156:Luciferases / Q000235:genetics*; D016366:Open Reading Frames; D010957:Plasmids; D014176:Protein Biosynthesis / Q000187:drug effects; D011691:Puromycin / Q000494:pharmacology; D012319:RNA Polymerase II / Q000378:metabolism; D012331:RNA, Fungal / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017384:Sequence Deletion; D014158:Transcription, Genetic
D012331:RNA, Fungal; D011691:Puromycin; D008156:Luciferases; D012319:RNA Polymerase II