Protein orientation in the Tat-TAR complex determined by psoralen photocross-linking.
Replication of 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'-end of all HIV mRNAs. We have used a new method based on psoralen photochemistry to identify a specific contact between a fragment of Tat protein (residues 38-72) and TAR RNA. We synthesized a 35-amino acid fragment containing arginine-rich RNA-binding domain of Tat (38-72), and replaced Arg57 with Cys to introduce a unique thiol group (-SH) in our model peptide. A psoralen derivative, which can react with thiol groups, was synthesized and used for specific chemical modification of Cys57-Tat-(38-72). We used this psoralen-Tat conjugate (psoralen-Cys57-Tat-(38-72)) to form a specific complex with TAR RNA. Upon near-ultraviolet irradiation (360 nm), this synthetic psoralen-peptide cross-linked to a single site in the TAR RNA sequence. The RNA-protein complex was purified and the cross-link site on TAR RNA was determined by RNA sequencing, which revealed that Cys57 of Tat is close to U31 of TAR RNA. Our results provide high-resolution proximity and orientation information about Tat-TAR complex. Such psoralen-peptide conjugates provide a new class of probes for sequence-specific protein-nucleic acid interactions and could be used to selectively control gene expression or to induce site-directed mutations.
Wang
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Department of Pharmacology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854, USA.
Wang
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Rana
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D000595:Amino Acid Sequence; D001120:Arginine; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D003545:Cysteine; D005363:Ficusin / Q000138:chemical synthesis; D011564:Furocoumarins / Q000138:chemical synthesis; D015696:Gene Products, tat / Q000737:chemistry* / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry; D011487:Protein Conformation; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism; D015533:Transcriptional Activation; D014466:Ultraviolet Rays; D054322:tat Gene Products, Human Immunodeficiency Virus
C100670:8-((3-iodoprop-1-yl)oxy)psoralen; D003432:Cross-Linking Reagents; D011564:Furocoumarins; D015696:Gene Products, tat; D010446:Peptide Fragments; D012333:RNA, Messenger; D012367:RNA, Viral; D054322:tat Gene Products, Human Immunodeficiency Virus; C021768:xanthotoxol; D001120:Arginine; D003545:Cysteine; D005363:Ficusin