Riboswitch as molecular tool for controlling artificially gene expression
Ellis O'Neill, Andrew Bassett, David Baulcombe, Alison G. Smith, and Michael Moulin
1) Department of Plant Sciences, University of Cambridge, Downing Street, CB2 3EA, UK
The ability to control the activity of a gene in a reversible and time dependent manner offers great advantages for studying gene function in vivo. We have optimized a new system for controlling gene expression in Chlamydomonas reinhardtii by using a thiamine pyrophosphate (TPP) riboswitch. Riboswitches are structures formed in the mRNA that bind specific small molecule ligands and alter gene expression. In Chlamydomonas, we have found TPP riboswitches in two vitamin B1 biosynthetic genes (1). Using a codon-optimized Gaussia princeps luciferase gene controlled by a constitutive promoter linked to a THI4 5' UTR riboswitch, we have now found that riboswitches are extremely efficient in manipulating transgene expression, and therefore protein level. Several constitutive promoters PSAD, RBCS and a chimeric promoter RBCS/HSP70 have been tested and results showed that the THI4 5' UTR riboswitch was compatible with all constitutive promoters. We observed that the system required 10 nM of thiamine in the external medium to repress gene expression within just 3 hours of incubation. When thiamine was removed from the medium, normal gene expression was recovered within 8 hours. When the RBCS/HSP70 promoter controlled the luciferase gene, the luciferase activity was rhythmic, the addition of the riboswitch downstream of the promoter did not affect the observed rhythm. Once thiamine was added to the medium, no luciferase activity was observed, the rhythm was later re-established when thiamine was removed from medium. Thus, TPP riboswitches provide an efficient means to control artificially and reversibly gene expression in algae. The use of thiamine, which is a non-toxic natural metabolite, avoids problems of contamination and metabolic perturbations that can occur in some inducible systems.

1) Croft MT, Moulin M, Webb ME, & Smith AG (2007) PNAS 104:20770-20775
e-mail address of presenting author: mm543@cam.ac.uk