A RNA polymerase III-based two-hybrid system to study RNA polymerase II transcriptional regulators

Journal of Molecular Biology
M C MarsolierA Sentenac

Abstract

In a previous study, we explored the mechanisms of SNR6 gene activation by grafting a heterologous DNA-binding domain, GAL4-(1-147), to various components of the yeast RNA polymerase III transcription system. Here, we demonstrate that a modified SNR6 gene harboring GAL4-binding sites (UAS(G)-SNR6) can be efficiently activated via an intervening, unrelated protein-protein interaction, thus laying the foundations of a RNA polymerase III-based two-hybrid system. In a model system, the interacting proteins recruiting TFIIIC to DNA were PRP21 and PRP9 or PRP21 and PRP11. Mutations affecting the interaction between PRP21 and PRP9, or PRP21 and PRP11 decreased UAS(G)-SNR6 activation level proportionally. RNA polymerase II transcriptional activators, like GAL4, VP16 or p53, fused to GAL4 DNA-binding domain, did not activate the UAS(G)-SNR6 gene. However, GAL4 strongly activated UAS(G)-SNR6 when GAL80, an interacting protein, was fused to TFIIIC. This result indicates that this two-hybrid system can be used to assess the interactions between RNA polymerase II regulatory proteins and their partners.

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Citations

Jul 9, 2005·Nucleic Acids Research·Michael PetrascheckAlcide Barberis
Jul 8, 2009·International Journal of Molecular Sciences·Anna BrücknerUwe Schlattner
Jul 12, 2001·Proceedings of the National Academy of Sciences of the United States of America·M HirstI Sadowski
Jul 12, 2002·Proteomics·Daniel AuerbachIgor Stagljar
Sep 1, 2000·FEBS Letters·P Legrain, L Selig
Jun 22, 2001·The Journal of Biological Chemistry·C ChangT Kodadek
Mar 21, 1998·Current Opinion in Biotechnology·R M Frederickson
Feb 18, 1999·Current Opinion in Chemical Biology·B L Drees

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