Characterization of recombinant HIV-1 Tat and its interaction with TAR RNA

Recombinant HIV-1 Tat (Tat 1-86) has been purified from the cytoplasmic fraction of Escherichia coli without the use of protein denaturants or chaotropic agents. Chloroquine-mediated uptake of the purified protein into cells resulted in transactivation of the HIV LTR promoter. Tat retains 1.64 mol o...

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Bibliographic Details
Published in:Biochemistry (Easton) 1992-12, Vol.31 (48), p.12062-12068
Main Authors: Slice, Lee W, Codner, Eileen, Antelman, Douglas, Holly, Maureen, Wegrzynski, Bogda, Wang, Jin, Toome, Voldemar, Hsu, Ming Chu, Nalin, Carlo M
Format: Article
Language:English
Subjects:
AIDS/HIV
Biological and medical sciences
Cell Line
Cloning, Molecular
Cross-Linking Reagents
Electrophoresis, Polyacrylamide Gel
Escherichia coli
Fundamental and applied biological sciences. Psychology
Gene Products, tat - chemistry
Gene Products, tat - genetics
Gene Products, tat - isolation & purification
Gene Products, tat - metabolism
Genetics
HIV Long Terminal Repeat - genetics
human immunodeficiency virus 1
Humans
Microbiology
Protein Binding
Protein Folding
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
RNA, Viral - metabolism
Transcriptional Activation
Virology
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Summary:Recombinant HIV-1 Tat (Tat 1-86) has been purified from the cytoplasmic fraction of Escherichia coli without the use of protein denaturants or chaotropic agents. Chloroquine-mediated uptake of the purified protein into cells resulted in transactivation of the HIV LTR promoter. Tat retains 1.64 mol of Zn2+/mol of protein by atomic absorption spectroscopy. Circular dichroism measurements indicated that the structure of recombinant Tat contains 15-20% alpha-helix. Filter binding assays showed that Tat binds to a 63-nucleotide target TAR RNA with a dissociation constant (Kd) of 10 nM at 25 degrees C, 0.05 M ionic strength, pH 7.5, in a 1:1 Tat-TAR RNA stoichiometry. Nonelectrostatic interactions provide the principal source of free energy of association. While the pH optimum occurs over a wide H+ concentration, the salt dependence of Kd indicates formation of a single ion pair. UV-induced protein-RNA cross-linking produced a labeled Tat-TAR RNA adduct, indicating that direct contact occurred between the Tat protein and TAR RNA.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi00163a014