Detection of specific human immunodeficiency virus type 1 Tat-TAR complexes in the presence of mild denaturing conditions.
Gene expression from the human immunodeficiency virus 1 (HIV-1) is greatly enhanced by binding of the virally encoded Tat protein to a 59-base RNA stem-loop structure, the Transactivation Responsive Element (TAR), located at the 5'-termini of all viral transcripts. This interaction was investigated in vitro using 32P-labelled TAR and highly purified Tat in which cysteine residues were blocked by sulpitolysis (S-Tat). It is shown that specific complex formation between S-Tat and TAR can occur in the presence of urea, with urea concentrations between 5 and 6 M causing an approximately two-fold increase in the level of binding. Two conditions favoring RNA secondary structure, low temperature (0 degree C) and the presence of divalent cations (Mg2+), diminished the level of specific binding. These observations suggest that the presence of mild denaturants promoted macromolecular refolding or rearrangement in a manner that increased the number of molecules available for binding, and present a general method for studying protein/RNA interactions where analysis has been obstructed by improper protein or RNA conformation.
Fong
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Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285-0438, USA. fong@mail.ncifcrf.gov
Smanik
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Thais
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Smith
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Jaskunas
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D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D015696:Gene Products, tat / Q000032:analysis* / Q000737:chemistry; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D012367:RNA, Viral; D014508:Urea; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D012367:RNA, Viral; D054322:tat Gene Products, Human Immunodeficiency Virus; D014508:Urea; D015500:Chloramphenicol O-Acetyltransferase