Self-association of Puralpha is mediated by RNA.
The 322 amino acid cellular protein, Puralpha, is a sequence-specific single-stranded DNA-binding protein implicated in control of transcription and replication. Previous studies have demonstrated that the interaction between Puralpha and its target DNA sequence results in the formation of multimeric complexes. In this study, we demonstrate that Puralpha 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 Puralpha, the domain which is important for the self-association of Puralpha 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 Puralpha. We have recently cloned a Puralpha associated-RNA, PU-RNA, and here we show that PU-RNA can specifically reconstitute the self-association of Puralpha. RNA not only mediates the self-association of Puralpha, but also modulates the ability of Puralpha to interact with its target DNA sequence. Electrophoretic mobility shift assays performed with and without RNase treatment demonstrate that RNA inhibits the interaction between Puralpha and its target DNA sequence. Moreover, we demonstrate that the self-association of Puralpha can be reconstituted by a specific oligonucleotide encompassing the Puralpha binding site. The implications of these findings with respect to Puralpha's role in transcription and replication are discussed.
Center for NeuroVirology and NeuroOncology, MCP Hahnemann University, Philadelphia, Pennsylvania 19102, USA.
G L
GL
Gallia
N
N
Darbinian
E M
EM
Johnson
K
K
Khalili
D000595:Amino Acid Sequence; D004247:DNA / Q000502:physiology; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D015696:Gene Products, tat / Q000378:metabolism; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D012313:RNA / Q000502:physiology*; D016601:RNA-Binding Proteins / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D015696:Gene Products, tat; C501130:PURA protein, human; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D012313:RNA; D004247:DNA