RNA-protein interactions in the Tat-trans-activation response element complex 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'-end of all HIV transcripts. We have used a site-specific cross-linking method based on 6-thioguanosine (6-thioG) photochemistry to determine the conformation of TAR RNA and its interaction with Tat protein under physiological conditions. Two different TAR RNA constructs with a single photoactive nucleoside (6-thioG) at position 21 or 26 were synthesized. Upon UV irradiation, 6-thioG at both positions formed interstrand covalent cross-links in TAR RNA. Determination of cross-link sites by RNA sequencing revealed that 6-thioG at position 21 contacts U42, while a 6-thioG at position 26 cross-links to C39. The addition of arginine did not alter the site of RNA-RNA cross-links; however, the yields of 6-thioG26-C39 cross-link were decreased. In the presence of a Tat fragment, Tat(38-72), UV irradiation of RNA modified with 6-thioG at position 21 resulted in RNA-protein cross-links but no RNA-RNA cross-links were observed. 6-thioG at position 26 formed both RNA-RNA and RNA-protein cross-links in the presence of Tat(38-72). Our results provide direct evidence that, during RNA-protein recognition, Tat is in close proximity to O6 of G21 and G26 in the major groove of TAR RNA.
Wang
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Department of Pharmacology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854, USA.
Rana
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D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism*; D003596:Cytosine / Q000378:metabolism; D015696:Gene Products, tat / Q000378:metabolism*; D016325:HIV Long Terminal Repeat / Q000528:radiation effects; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation / Q000528:radiation effects; D010777:Photochemistry; D012330:RNA, Double-Stranded / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism* / Q000528:radiation effects; D013866:Thioguanine / Q000378:metabolism; D014466:Ultraviolet Rays; D014498:Uracil / Q000378:metabolism
D000596:Amino Acids; D003432:Cross-Linking Reagents; D015696:Gene Products, tat; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D014498:Uracil; D003596:Cytosine; D013866:Thioguanine