Real-time kinetics of HIV-1 Rev-Rev response element interactions. Definition of minimal binding sites on RNA and protein and stoichiometric analysis.
The kinetics of interaction between the human immunodeficiency virus-1 Rev protein and its RNA target, Rev response element (RRE) RNA was determined in vitro using a biosensor technique. Our results showed that the primary Rev binding site is a core stem-loop RNA molecule of 30 nucleotides that bound Rev at a 1:1 ratio, whereas the 244-nucleotide full-length RRE bound four Rev monomers. At high Rev concentrations, additional binding of Rev to RRE was observed with ratios of more than 10:1. Because RRE mutants that lacked the core binding site and were inactive in vivo bound Rev nonspecifically at these concentrations, the real stoichiometric ratio of Rev-RRE is probably closer to 4:1. Binding affinity of Rev for RRE was approximately 10(-10) M, whereas the affinity for the core RNA was about 10(-11) M, the difference being due to the contribution of low affinity binding sites on the RRE. Mathematical analysis suggested cooperativity of Rev binding, probably mediated by the Rev oligomerization domains. C-terminal deletions of Rev had no effect on RRE binding, but truncation of the N terminus by as few as 11 residues significantly reduced binding specificity. This method was also useful to rapidly evaluate the potential of aminoglycoside antibiotics, to inhibit the Rev-RRE interaction.
Van Ryk
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Laboratory of Molecular Microbiology, NIAID, National Institutes of Health, Bethesda, Maryland 20892, USA.
Venkatesan
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D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D015694:Gene Products, rev / Q000737:chemistry*; D015752:Genes, env; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000737:chemistry; D020349:Surface Plasmon Resonance; D054321:rev Gene Products, Human Immunodeficiency Virus
D015694:Gene Products, rev; D009843:Oligoribonucleotides; D010446:Peptide Fragments; D016601:RNA-Binding Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus