Yeast two-hybrid screen for new transcriptional silencing proteins

Description

Abstract:
DNA codes the blueprint for every living organism, but the integrity of the DNA blueprint is threatened by corrupting transposon sequences. Transposons can proliferate and insert copies of themselves into new positions in DNA. The host cell protects itself against transposon expression and movement by targeting transposons for chemical modifications to DNA and associated histone proteins that silence transcription. Methylation of histone H3 at the lysine 9 position (H3K9me) is a conserved transposon silencing modification in eukaryotes. I am using the laboratory plant Arabidopsis thaliana to understand the structure and function of H3K9 methyltransferase proteins that catalyze the H3K9me modification. Three related H3 K9 methyltransferases—SUVH4, SUVH5, and SUVH6—are important factors in the Arabidopsis transcriptional silencing network. SUVH5 and SUVH6 are of particular interest because they each act preferentially on different transposon targets, but the mechanisms underlying their preferences remain to be elucidated. SUVH5 and SUVH6 have long amino terminal extensions beyond the parts of the protein that mediate histone methylation, including a conserved motif near the amino terminus. We hypothesize that the conserved amino terminal motif provides surfaces that interact with other proteins to help guide SUVH5 and SUVH6 to their preferred target transposons. To investigate this hypothesis, the Bender laboratory previously conducted a yeast two hybrid screen for proteins that can interact with the amino terminus of SUVH5 and SUVH6. This screen recovered a putative mRNA processing protein At4g36980. For my UTRA project, I designed, constructed, and tested mutations in the conserved amino terminal motifs of SUVH5 and SUVH6 to determine whether these mutations disrupt the yeast two hybrid interaction with At4g36980. I also designed, constructed and tested deletions in At4g36980 to determine whether a central serine-arginine (SR) rich region is the critical SUVH interaction motif.

Citation

Pallango, Savannah, "Yeast two-hybrid screen for new transcriptional silencing proteins" (2014). Summer Research Symposium. Brown Digital Repository. Brown University Library. https://doi.org/10.26300/p5ct-w290

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