Probing Tat peptide-TAR RNA interactions by psoralen photo-cross-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 site-specific cross-linking method based on psoralen photochemistry to determine the effect of core residues from the Tat sequence on the protein orientation in the Tat-TAR complex and on the specificity of Tat-TAR binding. We synthesized two Tat fragments, Tat(42-72) and Tat(37-72), and incorporated a psoralen-modified amino acid at position 41 during solid-phase assembly of the peptides. We used these psoralen-Tat conjugates to form specific complexes with TAR RNA. Upon near-ultraviolet irradiation (360 nm), psoralen-Asp41-Tat(37-72) 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 primer extension analysis, which revealed that Asp41 of Tat is close to U42 of the lower stem region of TAR RNA. Specificity of the RNA-peptide cross-linking reactions was determined by competition experiments. Our results show that the addition of only four residues (Cys37-Thr40) from the Tat core region significantly enhanced the specificity of the Tat peptide-TAR interactions without altering the site or chemical nature of the cross-link. These studies provide new insights into RNA-protein recognition that could be useful in designing peptidomimetics for RNA targeting. 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
Z
Z
Department of Pharmacology, UMDNJ-Robert Wood Johnson Medical School, and the Molecular Biology and Biochemistry Graduate Program at Rutgers State University, 675 Hoes Lane, Piscataway, New Jersey 08854, USA.
Shah
K
K
Rana
T M
TM
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000138:chemical synthesis / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism / Q000494:pharmacology; D005363:Ficusin / Q000378:metabolism*; D015696:Gene Products, tat / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D010777:Photochemistry; D011753:Pyrones / Q000378:metabolism; D012367:RNA, Viral / Q000138:chemical synthesis / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism / Q000494:pharmacology; D014529:Uridine / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus
D003432:Cross-Linking Reagents; D015696:Gene Products, tat; D010446:Peptide Fragments; D011753:Pyrones; D012367:RNA, Viral; D054322:tat Gene Products, Human Immunodeficiency Virus; D013912:Threonine; D001224:Aspartic Acid; D003545:Cysteine; D005363:Ficusin; D014529:Uridine