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. 2006 Aug 18;34(15):4278–4292. doi: 10.1093/nar/gkl499

Figure 1.

Figure 1

Specific Tat–TAR cross-link formation during transcription elongation. (A) Experimental design to incorporate a photoactive nucleoside into a nascent RNA chain during transcription elongation. Plasmid pWT2 harboring an inserted target sequence for triplex formation was linearized with restriction enzymes. A psoralen- and biotin-containing oligonucleotide was used to form triplex DNA. UV irradiation was then used to covalently cross-link psoralen to the template. Psoralen-cross-linked template was immobilized on streptavidin-conjugated magnetic beads and non-cross-linked DNA was washed away with buffer. Stepwise walking of RNA Pol II elongation complexes was used to incorporate the photoactive nucleoside 4-thioU into transcripts. Stalled TECs were incubated with Tat and UV irradiated to form the final cross-link product. (B) Site-specific photo-cross-linking of Tat and TAR RNA in stalled ternary complexes. Stepwise RNA Pol II walking was used to incorporate 4-thioU into TEC-associated transcripts at position 23. Tat was added to TECs stalled at C61 and UV irradiated (360 nm). The gel shows RNA from non-irradiated TECs (lane 1); UV-irradiated TECs (lane 2); TECs in the presence of 100 ng Tat but not UV irradiated (lane 3); TECs in the presence of Tat and UV irradiated (lane 4); TECs in the presence of Tat, UV irradiated, and digested with proteinase K at 37°C for 15 min (lane 5). RNA and RNA–protein cross-link are labeled as RNA and R-P XL, respectively. M is a marker lane. (C) Photo-cross-linking of TAR RNA in TECs stalled at C61 with Tat peptides. Lanes 1, 4 and 7 are non-irradiated control lanes with Tat peptides (amino acids 1–37), Tat peptide (amino acids 38–72), and full-length Tat, respectively. The gel shows RNA from TECs incubated with Tat peptide (amino acids 1–37) and UV irradiated (lanes 2 and 3); TECs incubated with Tat peptide (amino acids 38–72) and UV irradiated (lanes 5 and 6); TECs and full-length Tat and UV irradiated (lane 7). M is a marker lane (lane 8). (D) RNA–protein cross-linking between Tat and the nascent RNA chain in the TECs stalled at different positions. Elongation complexes containing 4-thioU at position 23 were stalled at G36, U42, U46, A51, C61 and C71. After incubating with Tat protein, photo-cross-linking reactions were performed and analyzed on denaturing gels as shown above. RNA–protein cross-linking yields were determined by phosphorimaging.