Real-time Kinetics of HIV-1 Rev-Rev Response Element Interactions
DEFINITION OF MINIMAL BINDING SITES ON RNA AND PROTEIN AND STOICHIOMETRIC ANALYSIS*
- From the Laboratory of Molecular Microbiology, NIAID, National Institutes of Health, Bethesda, Maryland 20892
Abstract
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 vivobound 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.
Footnotes
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↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed: NIAID, National Institutes of Health, Bldg. 10, Rm. 6A05, 9000 Rockville Pike, Bethesda, MD 20892-1576. Tel.: 301-496-6359; Fax: 301-402-4122; E-mail: aradhana{at}helix.nih.gov.
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↵2 M. Robinson, personal communication.
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↵3 K.-S. Jeong and S. Venkatesan, unpublished data.
- Abbreviations:
- HIV
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human immunodeficiency virus
- RRE
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Rev response element
- SPR
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surface plasmon resonance
- RU
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response unit(s)
- EMSA
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electrophoretic mobility shift assay
- T
-
truncated
- HH
-
hammerhead
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- Received January 6, 1999.
- Revision received April 7, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
