Abstract
We show that firefly luciferase is a stable protein when expressed at 25°C in Neurospora, which limits its use as transcription reporter. We created a short-lived luciferase by fusing a PEST signal to its C-terminus (LUC-PEST) and applied the LUC-PEST reporter system to record in vivo transcription dynamics associated with the Neurospora circadian clock and its blue-light photosensory system over the course of several days. We show that the tool is suitable to faithfully monitor rapid, but also subtle changes in transcription in a medium to high throughput format.
Highlights
Luciferase from Photinus pyralis is a highly sensitive reporter for monitoring gene expression activity in a variety of organisms [1,2,3,4,5]
We show that the destabilized LUCPEST protein (t1/2, 25 min) is a faithful reporter of promoter activity that readily uncovers rapid and considerably complex transcriptional dynamics
A PEST Signal Destabilizes Luciferase in Neurospora In order to decrease the half-life of firefly luciferase (LUC) a PEST-encoding DNA template [23] was codon optimized for Neurospora and fused to the 39 end of the codon-optimized luc ORF [19]
Summary
Luciferase from Photinus pyralis is a highly sensitive reporter for monitoring gene expression activity in a variety of organisms [1,2,3,4,5]. In Neurospora, the circadian transcription factor WHITE COLLAR COMPLEX (WCC) controls rhythmic expression of FREQUENCY (FRQ), which in turn inhibits the WCC in a negative feedback loop [11,12,13,14]. In light-dark cycles the activity of the WCC is attenuated by the LOV-photoreceptor VIVID [16,17,18], which directly interferes with light-dependent dimerization of the WCC via LOV-LOV interactions [15]. These feedback loops support rhythmic transcription of frq and other clock-controlled genes in constant darkness and in light-dark cycles
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