Chemical Cross-Linking of the Human Immunodeficiency Virus Type 1 Tat Protein to Synthetic Models of the RNA Recognition Sequence TAR Containing Site-Specific Trisubstituted Pyrophosphate Analogues†
Identifieur interne : 000877 ( Istex/Curation ); précédent : 000876; suivant : 000878Chemical Cross-Linking of the Human Immunodeficiency Virus Type 1 Tat Protein to Synthetic Models of the RNA Recognition Sequence TAR Containing Site-Specific Trisubstituted Pyrophosphate Analogues†
Auteurs : Nikolai A. Naryshkin [Russie] ; Mark A. Farrow [Russie] ; Marina G. Ivanovskaya [Russie] ; Tanya S. Oretskaya [Russie] ; Zoe A. Shabarova [Russie] ; Michael J. Gait [Russie, Royaume-Uni]Source :
- Biochemistry [ 0006-2960 ] ; 1997.
Abstract
A chemical ligation procedure has been developed for the synthesis of oligoribonucleotides carrying a trisubstituted pyrophosphate (tsp) linkage in place of a single phosphodiester. Good yields of tsp were obtained when a single 2‘-deoxynucleoside 5‘ to the tsp was used in the ligation reaction. A tsp linkage was found to be reasonably stable to hydrolysis but cleaved by treatment with ethylenediamine or lysine to give phosphoamidate adducts. A model human immunodeficiency virus type 1 (HIV-1) TAR RNA duplex containing an activated tsp was able to bind to HIV-1 Tat protein with only 3-fold reduced affinity and to a Tat peptide (residues 37−72) with identical affinity compared to that of an unmodified duplex. Tsps incorporated at sites previously identified as being in close proximity to Tat protein were able to cross-link to Tat peptide (37−72) to form a covalent phosphoamidate conjugate. Endopeptidase cleavage followed by MALDI-TOF (matrix-assisted laser desorption ionization time of flight) mass spectrometric analysis provided strong evidence that a TAR duplex containing a tsp replacing the phosphate at 38−39 had reacted specifically with Lys51 in the basic region of Tat peptide (37−72). The new chemical cross-linking method may be generally useful for identifying lysines in close proximity to phosphates in basic RNA-binding domains of proteins.
Url:
DOI: 10.1021/bi962789p
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<front><div type="abstract">A chemical ligation procedure has been developed for the synthesis of oligoribonucleotides carrying a trisubstituted pyrophosphate (tsp) linkage in place of a single phosphodiester. Good yields of tsp were obtained when a single 2‘-deoxynucleoside 5‘ to the tsp was used in the ligation reaction. A tsp linkage was found to be reasonably stable to hydrolysis but cleaved by treatment with ethylenediamine or lysine to give phosphoamidate adducts. A model human immunodeficiency virus type 1 (HIV-1) TAR RNA duplex containing an activated tsp was able to bind to HIV-1 Tat protein with only 3-fold reduced affinity and to a Tat peptide (residues 37−72) with identical affinity compared to that of an unmodified duplex. Tsps incorporated at sites previously identified as being in close proximity to Tat protein were able to cross-link to Tat peptide (37−72) to form a covalent phosphoamidate conjugate. Endopeptidase cleavage followed by MALDI-TOF (matrix-assisted laser desorption ionization time of flight) mass spectrometric analysis provided strong evidence that a TAR duplex containing a tsp replacing the phosphate at 38−39 had reacted specifically with Lys51 in the basic region of Tat peptide (37−72). The new chemical cross-linking method may be generally useful for identifying lysines in close proximity to phosphates in basic RNA-binding domains of proteins.</div>
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