A modified yeast one-hybrid (MY1H) system has been developed for in vivo investigation of simultaneous protein-protein and protein:DNA interactions. The traditional yeast one-hybrid assay (Y1H) permits examination of one expressed protein targeting one DNA site, whereas our MY1H allows coexpression of two different proteins and examination of their activity at the DNA target. This single-plasmid based MY1H was validated by use of the DNA-binding protein p53 and its inhibitory partners, large T antigen (LTAg) and 53BP2. The MY1H system could be used to examine proteins that contribute inhibitory, repressive, coactivational or bridging functions to the protein under investigation, as well as potential extension toward library screening for identification of novel accessory proteins.
After development and validation of the MY1H with the p53/LTAg/53BP2 system, we applied the MY1H system to investigate the DNA binding activities of heterodimeric proteins, the bHLH/PAS domains of AhR and Arnt that target the xenobiotic response element (XRE). The AhR/Arnt:XRE interaction, which served as our positive control for heterodimeric protein binding of the XRE DNA site, showed negative signals in initial MY1H experiments. These false negative observations were turned into true positives by increasing the number of DNA target sites upstream of the reporter genes and increasing the number of activator domains fused to the two monomers. This methodology may help trouble-shooting false negatives stemming from unproductive transcription in yeast genetic assays, which can be a common problem.
In the study of XRE-binding proteins, two bHLHZ-like hybrid proteins, AhRJunD and ArntFos were designed and coexpressed in the MY1H and yeast two-hybrid (Y2H) systems; these proteins comprise the bHLH domains of AhR and Arnt fused to the leucine zipper (LZ) elements from bZIP proteins JunD and Fos, respectively. The in vivo assays revealed that in the absence of the XRE DNA site, heterodimers and homodimers formed, but in the presence of the nonpalindromic XRE, only heterodimers bound to the XRE and activated reporter transcription. The present results provide valuable information on DNA-mediated protein heterodimerization and specific DNA binding, as well as the relationship between protein structure and DNA-binding function.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/19337 |
| Date | 18 March 2010 |
| Creators | Chen, Gang |
| Contributors | Shin, Jumi A. |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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