The use of altered specificity mutants to probe specific protein-protein interactions involved in the activation of GATA-1 target genes

John D. Crispino*, Stuart H. Orkin

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

Abstract

With the increasing popularity of the yeast two-hybrid screen, a large number of protein-protein interactions have been identified. In many cases, single proteins have been found to associate with a large number of cofactors. For example, the hematopoietic transcription factor GATA-1 interacts with a multitude of other nuclear proteins, including Friend of GATA-1 (FOG-1), EKLF, CBP/p300, and Lmo2. Furthermore, p300, besides associating with GATA-1, interacts with at least seven other hematopoietic transcription factors. Despite the numerous pairwise and higher-order interactions identified, assessment of their contribution to transcriptional control has been lacking. Here we describe a strategy that can be applied to assess the functional relevance of any protein-protein association. This approach involves the creation of altered specificity mutants though the use of a combination of two yeast two-hybrid screens. Once altered specificity factors are obtained, a researcher can then proceed to functional assays that address the role of a specific protein-protein interaction.

Original languageEnglish (US)
Pages (from-to)84-92
Number of pages9
JournalMethods
Volume26
Issue number1
DOIs
StatePublished - 2002

Keywords

  • Altered specificity mutants
  • Transcription factors
  • Yeast two-hybrid

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)

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