On-off controllable RNA hybrid expression vector for yeast three-hybrid system.
The yeast three-hybrid system (Y3H), a powerful method for identifying RNA-binding proteins, still suffers from many false positives, due mostly to RNA-independent interactions. In this study, we attempted to efficiently identify false positives by introducing a tetracycline operator (tetO) motif into the RPR1 promoter of an RNA hybrid expression vector. We successfully developed a tight tetracycline-regulatable RPR1 promoter variant containing a single tetO motif between the transcription start site and the A-box sequence of the RPR1 promoter. Expression from this tetracycline-regulatable RPR1 promoter in the presence of tetracycline-response transcription activator (tTA) was positively controlled by doxycycline (Dox), a derivative of tetracycline. This on-off control runs opposite to the general knowledge that Dox negatively regulates tTA. This positively controlled RPR1 promoter system can therefore efficiently eliminate RNA-independent false positives commonly observed in the Y3H system by directly monitoring RNA hybrid expression.
Bak
Geunu
G
Department of Chemistry, KAIST, Daejeon 305-701, Korea.
Hwang
Se Won
SW
Ko
Yerim
Y
Lee
Jungmin
J
Kim
Youngmi
Y
Kim
Kyunghwan
K
Hong
Soon Kang
SK
Lee
Younghoon
Y
D001483:Base Sequence; D004318:Doxycycline / Q000494:pharmacology; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D015534:Trans-Activators / Q000187:drug effects / Q000235:genetics; D020798:Two-Hybrid System Techniques
D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D004318:Doxycycline