Optimisation of the Schizosaccharomyces pombe urg1 Expression System

Abstract

The ability to study protein function in vivo often relies on systems that regulate the presence and absence of the protein of interest. Two limitations for previously described transcriptional control systems that are used to regulate protein expression in fission yeast are: the time taken for inducing conditions to initiate transcription and the ability to achieve very low basal transcription in the “OFF-state”. In previous work, we described a Cre recombination-mediated system that allows the rapid and efficient regulation of any gene of interest by the urg1 promoter, which has a dynamic range of approximately 75-fold and which is induced within 30-60 minutes of uracil addition. In this report we describe easy-to-use and versatile modules that can be exploited to significantly tune down Purg1 “OFF-levels” while maintaining an equivalent dynamic range. We also provide plasmids and tools for combining Purg1 transcriptional control with the auxin degron tag to help maintain a null-like phenotype. We demonstrate the utility of this system by improved regulation of HO-dependent site-specific DSB formation, by the regulation Rtf1-dependent replication fork arrest and by controlling Rhp18Rad18-dependent post replication repair.

An Estradiol-Inducible Promoter Enables Fast, Graduated Control of Gene Expression in Fission Yeast

“…However, the absolute level of expression from any promoter will depend on the stability of the target gene's message and protein product. For applications that require lower basal expression levels, gene expression could be modified by adding mRNA or protein destabilizing elements (Voon et al, 2005), as has been done with the urg1 promoter (Watson et al, 2013).…”

“…Nonetheless, mitochondrial toxicity of tetracycline remains a concern (Moullan et al, 2015). The endogenous urg1 promoter has been shown to have favourable induction and repression kinetics (Watt et al, 2008), but it suffers from the disadvantages of being regulated by uracil (which requires growth in defined media and has other transcriptional effects), working best only at its endogenous locus (Watson et al, 2013), and not having a well-characterized dose response.…”

“…Fission Yeast Z3EV Promoter experimental flexibility. Other S. pombe promoter systems have been modified to produce a series of promoters of varying strengths (Basi et al, 1993;Watson et al, 2013). However, this approach allows access to only a fixed number of expression levels and does not allow flexible control of expression within one strain.…”

“…However, this approach allows access to only a fixed number of expression levels and does not allow flexible control of expression within one strain. The linear dose-response of Z 3 EV, in combination with the use of mRNA and protein destabilization elements to modify the absolute range of Z 3 EVpr-driven expression for any particular target gene, will afford a wide range of expression levels for any target protein (Voon et al, 2005;Watson et al, 2013).…”

A novel protein, Rsf1/Pxd1, is critical for the single‐strand annealing pathway of double‐strand break repair in Schizosaccharomyces pombe

“…To address the SSA pathway in fission yeast, we and other groups established a chromosome‐based SSA repair system in S. pombe (Osman et al ., ; Blaikley et al ., ). Recently, Antony M. Carr's group established a novel S. pombe SSA assay (Watson et al ., 2011; 2013). In their system, HO endonuclease expression is controlled by a Purg1 promoter, which is induced by uracil.…”