Figure 6

Influence of incubation temperature and stem B truncation on lentiviral leader RNA tight dimerization. The RNAs tested in panel A include the entire stem B (1–444 HIV-2, 1–441 SIV, and 1–277 HIV-1) and in panel B exclude the 3' side of stem B (1–437 HIV-2, 1–435 SIV, and 1–272 HIV-1) (see Figure 5, top panel, for structural details). A and B. All RNAs were individually incubated at the indicated temperatures (24°, 37°, 40°, 45°, 50°, 55°, and 60°C) for 30 min in their corresponding dimer buffer and subjected to electrophoresis on TBE agarose gel run at 26°C. Lane 1 represents monomeric RNA that was denatured at 90°C, then quenched on ice immediately prior to loading. C. A mutated 1–444 HIV-2 RNA with an extended SL1 was incubated at the indicated temperatures (24°, 37°, 40°, 45°, 50°, 55°, 60°, and 70°C) for 30 min in the dimer buffer H and subjected to electrophoresis on TBE agarose gel run at 26°C. The extension of SL1 is caused by the substitution of nts G⁴³⁷-U⁴³⁸ by the sequence 5'-CUUUCUA-3' (right panel, boxed letters). This mutation doubles the stability of the SL1 region, from -18.2 to -36.4 kcal/mol (ΔGs of wild type and mutated 1–444 HIV-2 RNAs, respectively).