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 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.
Slice
L W
LW
Department of Virology, Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey 07110.
Codner
E
E
Antelman
D
D
Holly
M
M
Wegrzynski
B
B
Wang
J
J
Toome
V
V
Hsu
M C
MC
Nalin
C M
CM
D002460:Cell Line; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015696:Gene Products, tat / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D006801:Humans; D011485:Protein Binding; D017510:Protein Folding; D012367:RNA, Viral / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015533:Transcriptional Activation
D003432:Cross-Linking Reagents; D015696:Gene Products, tat; D012367:RNA, Viral; D011994:Recombinant Proteins