A RNA polymerase III-based two-hybrid system to study RNA polymerase II transcriptional regulators.
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.
Marsolier
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MC
Service de Biochimie et de Génétique Moléculaire, CEA-Saclay, Gir-sur-Yvette, France.
Prioleau
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MN
Sentenac
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A
D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D011485:Protein Binding; D012319:RNA Polymerase II / Q000378:metabolism*; D012320:RNA Polymerase III / Q000378:metabolism*; D012343:RNA, Transfer / Q000235:genetics*; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000378:metabolism*; D019474:Transcription Factors, TFIII; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D000098832:Transcription Factor TFIIIC
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; C055002:GAL80 protein, S cerevisiae; C082350:PRP21 protein, S cerevisiae; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D019474:Transcription Factors, TFIII; D000098832:Transcription Factor TFIIIC; D012343:RNA, Transfer; D012319:RNA Polymerase II; D012320:RNA Polymerase III