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KMID : 0624620100430020110
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BMB Reports
2010 Volume.43 No. 2 p.110 ~ p.114
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On-off controllable RNA hybrid expression vector for yeast three-hybrid system
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Bak Geu-Nu
Ko Ye-Rim
Lee Jung-Min
Kim Young-Mi
Kim Kyung-Hwan
Lee Young-Hoon
Hwang Se-Won
Hong Soon-Kang
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Abstract
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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.
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KEYWORD
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RNA hybrid expression vector, RPR1 promoter, Tetracycline regulation, Yeast three-hybrid system
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