In recent years, through parallel advances in microscopy, fluorescent probe development, and computational modeling, it has become possible to describe gene expression in a fundamentally different way: one can now directly observe single molecules of RNA in living and fixed cells using the fluorescence microscope. In this talk, I will describe the use of this single-molecule approach to study transcription kinetics of the GAL10 locus in budding yeast, which is regulated by sugar availability. Since the discovery that eukaryotic transcription is pervasive, the question is whether non-coding (nc) transcription is transcriptional noise or has a functional role. By visualizing both coding and non-coding transcription at the GAL cluster in living yeast cells, we show that transcription of the same ncRNA can switch between functional and non-functional roles in different conditions. We show that GAL10 ncRNA, transcribed from the antisense strand is only transiently present at the locus when it is being transcribed and is not maintained on chromatin to form R-loops. During galactose induction, GAL10 transcription occurs in bursts of high activity followed by periods of inactivity. During these activating conditions, GAL10 transcription is unaffected by spurious transcription of GAL10 ncRNA, even when they occur almost simultaneously at the same locus. In contrast, in conditions where GAL10 is ‘off’, GAL10 ncRNA transcription is important to prevent transcriptional leakage of GAL1 and GAL10. The leakage is independent of the Gal4p transcription factor and not abolished by glucose repression, indicating that ncRNA transcription forms an additional layer of repression. Our results demonstrate that ncRNA transcription is necessary to maintain transcription in an off-state, but that ncRNA transcription per se is not repressive and can become non-functional during activation.
Lunch @ 11:45, talk @ 12pm.