Conditional gene expression by controlling translation with tetracycline-binding aptamers.
We present a conditional gene expression system in Saccharomyces cerevisiae which exploits direct RNA-metabolite interactions as a mechanism of genetic control. We inserted preselected tetracycline (tc) binding aptamers into the 5'-UTR of a GFP encoding mRNA. While aptamer insertion generally reduces GFP expression, one group of aptamers displayed an additional, up to 6-fold, decrease in fluorescence upon tc addition. Regulation is observed for aptamers inserted cap-proximal or near the start codon, but is more pronounced from the latter position. Increasing the thermodynamic stability of the aptamer augments regulation but reduces expression of GFP. Decreasing the stability leads to the opposite effect. We defined nucleotides which influence the regulatory properties of the aptamer. Exchanging a nucleotide probably involved in tc binding only influences regulation, while mutations at another position alter expression in the absence of tc, without affecting regulation. Thus, we have developed and characterized a regulatory system which is easy to establish and controlled by a non-toxic, small ligand with good cell permeability.
Suess
Beatrix
B
Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstrasse 5, 91058 Erlangen, Germany. bsuess@biologie.uni-erlangen.de
Hanson
Shane
S
Berens
Christian
C
Fink
Barbara
B
Schroeder
Renée
R
Hillen
Wolfgang
W
D020121:5' Untranslated Regions / Q000235:genetics; D001483:Base Sequence; D005786:Gene Expression Regulation / Q000187:drug effects; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D016254:Mutagenesis, Insertional; D009154:Mutation; D009843:Oligoribonucleotides / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D013752:Tetracycline / Q000378:metabolism* / Q000494:pharmacology; D014170:Transformation, Genetic
D020121:5' Untranslated Regions; D008164:Luminescent Proteins; D009843:Oligoribonucleotides; D049452:Green Fluorescent Proteins; D013752:Tetracycline