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.
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.
Naryshkin
N A
NA
Laboratory of Molecular Biology, Medical Research Council, Cambridge, U.K.
Farrow
M A
MA
Ivanovskaya
M G
MG
Oretskaya
T S
TS
Shabarova
Z A
ZA
Gait
M J
MJ
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D011756:Diphosphates / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015696:Gene Products, tat / Q000138:chemical synthesis / Q000378:metabolism*; D016325:HIV Long Terminal Repeat; D015497:HIV-1; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013698:Templates, Genetic; D054322:tat Gene Products, Human Immunodeficiency Virus
D003432:Cross-Linking Reagents; D011756:Diphosphates; D015696:Gene Products, tat; D009841:Oligonucleotides; D010446:Peptide Fragments; D012367:RNA, Viral; D054322:tat Gene Products, Human Immunodeficiency Virus