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.