Acquired mutations in TAR restore secondary structure but not Tat binding. In vitro transcribed TAR RNA corresponding to the wild-type SIVmac239 (TARwt), SIV-rtTA (TARm) and the evolved +21G-A, +63A-G and +78C-U variants was denatured by heat, renatured in the presence of MgCl2 and analyzed on a denaturing gel (A) and on a non-denaturing gel (B). Under these non-denaturing conditions, branched hairpin (BH) RNA conformers migrate slower than extended hairpin (EH) molecules . (C) Binding of SIV Tat to TAR was analyzed in an Electrophoretic Mobility Shift Assay (EMSA). TAR RNA was incubated with 0 or 100 ng Tat protein (indicated with - and +, respectively) and analyzed on a non-denaturing gel. The position of unbound TAR RNA and TAR-Tat complex is indicated.