NIH Mock Proposal
1. Specific Aims
2. Background and Significance
3. Research Design and Methods
   1. Yeast Genetics
   2. Immunopre- cipitation
   3. Yeast Two-Hybrid
   4. Future Experiments
4. References

3.3 Determination of protein interactions with frataxin through the yeast two-hybrid system

3.3.1 Yeast two-hybrid

In the case that frataxin may only interact with low affinity to any other protein, detection may be difficult or impossible with immunoprecipitation. The yeast two-hybrid system would then be employed as it can detect strong and weak affinity interactions. The two-hybrid system takes advantage of the modular nature of eukaryotic transcription activators; many have at least two distinct functional domains, one that directs binding to specific DNA sequences and one that activates transcription.²⁰ The DNA-binding and activation domains need not be covalently bonded for activation to occur.²⁰ The yeast two-hybrid system works by allowing two interacting proteins to bring a transcription activation domain in close proximity to a DNA binding domain, allowing transcription and thus expression of a reporter gene.²¹ The power of the yeast two-hybrid system has been improved by the addition of more reporter genes, eliminating much of the time spent on screening false positives. To set up the experiment, YFH1 can be fused to the LexA binding domain, and DNA from a yeast genomic library (readily available from many biotech companies) will be fused to the B42 transcriptional activator domain. Interaction of YFH1 with another protein will allow transcription of leu2 and lacZ, downstream reporters under control of the LexA activator. The sensitivity of the system can be adjusted by varying the amount of LexA operators in the plasmid used; for example, some plasmids may have two operators while others have eight.²¹ Transcription of the leu2 gene will allow yeast colonies to grow on leucine deficient media, and transcription of lacZ can be visualized as blue colonies will form on plates containing X-Gal. After proteins are identified in this fashion, the clones can be isolated, and the interaction can be tested physiologically by ensuring that the putative interacting protein also co-localizes to the mitochondria. This can be accomplished by tagging the proteins with fluorescent markers as previously described.^8,14 Once it is determined that the proteins actually interact in vivo, the necessary amino acid residues will then be determined. This can also be determined by the two-hybrid system by constructing YFH1 deletions and determining which regions are necessary for reporter gene activation. The area of interaction can further be defined by site-directed mutagenesis to find specific amino acids necessary for the interaction.

3.3.2 Interpretation

The yeast two-hybrid system is used as an alternate method because of the time involved in initial set up and screening. The yeast two-hybrid system has been known to generate many false positives, so all positives must be rigorously rescreened. The use of multiple reporter genes should eliminate some of the work. Additionally, sometimes an interaction is missed and false negative results are obtained. Careful use of controls and following troubleshooting guides published by Origene and others can alleviate most of the problems. After it is determined that activation is indeed produced by a cDNA, the physiological relevance can be determined by co-localization experiments. If positive results are obtained at this point, it is likely that an actual interaction has been identified. Identification of the specific residues involved in interaction provides the final confirmation.

3.3.3 Protocol

The methodology of the yeast two-hybrid system has been covered extensively in the literature. Many protocols exist, and for this experiment a protocol and kit designed by Origene will be used. Origene's system is based upon the protocol used by the Brent lab (Harvard).²⁰ This system was chosen because it utilizes LexA/B42, and a compatible yeast cDNA library is readily available. LexA/B42 are not found naturally in yeast, and this will help reduce the amount of false positives by eliminating some endogenous interactions that could occur with the Gal4 system. The system will be initially set up using plasmids that can detect weak interactions since no proteins were detected by immunoprecipitation. If too many false positives result, plasmids that detect stronger interactions will be used. Details of the strains are available from Origene and the Brent lab.^20,22 YFH1 must be inserted into the plasmids in frame, so standard PCR techniques to add restriction sites will be employed. Additionally, YFH1 has a mitochondrial targeting sequence at its N-terminus that is cleaved during maturation of the protein.¹³ This sequence could interfere with localization of the LexA-YFH1 fusion protein to the nucleus, so the first 40 amino acids will not be included in the fusion product. Appropriate controls and screens for false positives will be used throughout the procedure as outlined in published protocols.

Next Section: 3.4 Future Experimentation

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