Proximity of a Tat peptide to the HIV-1 TAR RNA loop region determined by site-specific photo-cross-linking.
Transcriptional regulation in human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5'-ends of all HIV-1 mRNAs. We have used a site-specific cross-linking method based on 4-thio-uracil (4-thioU) photochemistry to determine the interactions of a Tat peptide, Tat(38-72), with the loop region of TAR RNA under physiological conditions. A TAR RNA construct with a single 4-thioU residue at positions U31 in the loop sequence was synthesized by chemical methods. Upon UV irradiation, 4-thioU at U31 formed a covalent cross-link with the Tat peptide. We did not observe any RNA-RNA cross-link formation. Competition experiments revealed that a specific RNA-protein complex formation was necessary for the RNA-protein cross-linking reaction. Our results demonstrate that, during RNA-protein recognition, the Tat peptide is located in close proximity to O4 of U31 in the TAR RNA loop sequence.
Wang
Z
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Department of Pharmacology, Robert Wood Johnson Medical School-UMDNJ, and Molecular Biosciences Graduate Program at Rutgers University, 675 Hoes Lane, Piscataway, New Jersey 08854, USA.
Huq
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Rana
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D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry; D015696:Gene Products, tat / Q000378:metabolism*; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010446:Peptide Fragments / Q000378:metabolism; D010777:Photochemistry; D011485:Protein Binding; D012367:RNA, Viral / Q000378:metabolism*; D013889:Thiouracil / Q000031:analogs & derivatives / Q000737:chemistry; D013891:Thiouridine / Q000737:chemistry; D054322:tat Gene Products, Human Immunodeficiency Virus
D003432:Cross-Linking Reagents; D015696:Gene Products, tat; D010446:Peptide Fragments; D012367:RNA, Viral; D054322:tat Gene Products, Human Immunodeficiency Virus; D013891:Thiouridine; C114719:4-thiouracil; D013889:Thiouracil