Self-association of Purα is mediated by RNA

Gary L. Gallia, Nune Darbinian, Edward M. Johnson, Kamel Khalili

Research output: Contribution to journalArticlepeer-review

Abstract

The 322 amino acid cellular protein, Purα, is a sequence-specific single-stranded DNA-binding protein implicated in control of transcription and replication. Previous studies have demonstrated that the interaction between Purα and its target DNA sequence results in the formation of multimeric complexes. In this study, we demonstrate that Purα can self- associate in the absence of DNA. This self-association, while independent of DNA, is mediated by RNA. Through in vitro studies with bacterially expressed glutathione S-transferase fusion proteins, and the synthetic peptides corresponding to various central regions of Purα, the domain which is important for the self-association of Purα is localized to acidic leucine- rich repeats. Interestingly, these repeats have previously been shown to interact with the human immunodeficiency virus 1 (HIV-1) Tat protein and in this study we demonstrate that Tat is able to disrupt the self-association of Purα. We have recently cloned a Purα associated-RNA, PU-RNA, and here we show that PU-RNA can specifically reconstitute the self-association of Purα. RNA not only mediates the self-association of Purα, but also modulates the ability of Purα to interact with its target DNA sequence. Electrophoretic mobility shift assays performed with and without RNase treatment demonstrate that RNA inhibits the interaction between Purα and its target DNA sequence. Moreover, we demonstrate that the self-association of Purα can be reconstituted by a specific oligonucleotide encompassing the Purα binding site. The implications of these findings with respect to Purα's role in transcription and replication are discussed.

Original languageEnglish (US)
Pages (from-to)334-348
Number of pages15
JournalJournal of cellular biochemistry
Volume74
Issue number3
DOIs
StatePublished - Sep 1 1999
Externally publishedYes

Keywords

  • HIV-1 tat protein
  • Purα
  • RNA
  • Replication
  • Self-association
  • Transcription

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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