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Norbert Lehming Staatsexamen
(BSc Hons equivalent), PhD,
Habilitation (2000)
Assistant Professor
Tel: +65 6516 3499
Email: micln@nus.edu.sg |
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The regulation of transcription is the focus of interest. Transcription is the basis for the realization
of genetic information, and its regulation is of vital interest for all organisms. Transcriptional
regulation is achieved by protein interactions between DNA-bound transcription factors.
Split-Ub, an alternative two-hybrid system, is used to analyze these protein interactions inside
living cells. Split-Ub is based upon conditional degradation: A protein of interest is fused
to the C-terminal half of ubiquitin, which is C-terminally extended by a reporter protein like
RUra3p, RGpt2p, or RGFP. In the reporter protein, the fi rst amino acid has been replaced
by arginine, an instable residue in the N-end rule pathway of protein degradation.
A second
protein of interest can be tested for interaction with the first protein by fusing it to the
N-terminal half of ubiquitin. The local concentrations of the two halves of ubiquitin will only
be high enough to allow the formation of a native-like ubiquitin if the two proteins interact
inside the cell. The native-like ubiquitin is cleaved by the ubiquitin-specifi c proteases, and
the free reporter is degraded by the enzymes of the N-end rule. Therefore, yeast cells become
resistant to 5-fl uoro orotic acid, a drug counterselecting Ura3p, and human tissue culture
cells become resistant to 6-thioguanine, a drug counterselecting Gpt2p, upon interaction
of the two proteins inside these cells. The GFP reporter can be used in any organism.
The system has been used to screen libraries in order to identify new interaction partners for
transcriptional activators and repressors. |
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Xiaowei Xue and N Lehming (2008) Nhp6p and Med3p regulate gene expression by controlling the local subunit composition of RNA polymerase II. J. Mol. Biol. 379:212-230
Lim MK, Tang V, Le Saux A, Schuller J, Bongards C and Lehming N (2007) Ga11 1p mpensates transcriptional activator deletions via Taf14p. J. Mol. Biol. 374:9-23
Chew, BS and N Lehming (2007) TFIIB/SUA7 (E202G) is an allele-specific suppressor of TBP1 (E186D). Biochemical. J. 406:265-271
Bongards C, Chew BS and Lehming N (2003) The TATA-binding protein is not an essential target of the transcriptional activators Gal4p and Gcn4p in Saccharomyces cerevisiae. Biochemical. J. 370:141-147
Ansari AZ, Koh SS, Zaman Z, Bongards C, Lehming N, Young RA and Ptashne M (2002) Transcriptional activating regions target a cyclin-dependent kinase. Proc. Natl. Acad. Sci. USA. 99:14706-14709
Pätzold, AJ and Lehming N (2001) Why Ppr1p is a weak activator of transcription. FEBS Lett. 494:64-68
Gromöller A and Lehming N (2000) Srb7p is a physical and physiological target of Tup1p. EMBO J. 19:6845-6852
Rojo-Niersbach, E, Morley, D, Heck, S, and Lehming N (2000) A new method for the selection of protein interactions in mammalian cells. Biochemical J. 348:585-590
Laser H, Bongards C, Schüller J, Heck S, Johnsson N, and Lehming N (2000) A new screen for protein interactions reveals that the Saccharomyces cerevisiae high mobility group proteins Nhp6A/B are involved in the regulation of the GAL1 promoter. Proc. Natl. Acad. Sci. USA 97:13732-13737 |
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