translation agency

Detection of specific human immunodeficiency virus type 1 Tat-TAR complexes in the presence of mild denaturing conditions.
Fong SE; Smanik P; Thais T; Smith MC; Jaskunas SR; Lilly Research
November 30, 1997
J Virol Methods. 1997 Jun;66(1):91-101. Unique Identifier : AIDSLINE

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.

*Gene Products, tat/ANALYSIS *HIV Long Terminal Repeat *HIV-1/GENETICS *HIV-1/METABOLISM