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 : 003C84 ( Main/Exploration ); précédent : 003C83; suivant : 003C85Chemical 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
Affiliations:
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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></front></TEI><affiliations><list><country><li>Royaume-Uni</li><li>Russie</li></country><region><li>District fédéral central</li></region><settlement><li>Moscou</li></settlement><orgName><li>Université d'État de Moscou</li></orgName></list><tree><country name="Russie"><region name="District fédéral central"><name sortKey="Naryshkin, Nikolai A" sort="Naryshkin, Nikolai A" uniqKey="Naryshkin N" first="Nikolai A." last="Naryshkin">Nikolai A. Naryshkin</name></region><name sortKey="Farrow, Mark A" sort="Farrow, Mark A" uniqKey="Farrow M" first="Mark A." last="Farrow">Mark A. Farrow</name><name sortKey="Gait, Michael J" sort="Gait, Michael J" uniqKey="Gait M" first="Michael J." last="Gait">Michael J. Gait</name><name sortKey="Ivanovskaya, Marina G" sort="Ivanovskaya, Marina G" uniqKey="Ivanovskaya M" first="Marina G." last="Ivanovskaya">Marina G. Ivanovskaya</name><name sortKey="Oretskaya, Tanya S" sort="Oretskaya, Tanya S" uniqKey="Oretskaya T" first="Tanya S." last="Oretskaya">Tanya S. Oretskaya</name><name sortKey="Shabarova, Zoe A" sort="Shabarova, Zoe A" uniqKey="Shabarova Z" first="Zoe A." last="Shabarova">Zoe A. Shabarova</name></country><country name="Royaume-Uni"><noRegion><name sortKey="Gait, Michael J" sort="Gait, Michael J" uniqKey="Gait M" first="Michael J." last="Gait">Michael J. Gait</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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