RNAs undergo conformational adaptation upon binding to proteins and small molecules to optimize inter-molecular interactions. The thermodynamic propensity of an RNA to adopt a bound conformational ensemble is an important determinant of the overall thermodynamic binding affinity. However, little is known regarding this energetic cost because it requires measuring the relative populations of low-abundance short-lived bound conformations in the apo-ensemble that may not be detectable using conventional biophysical methods. Here, using NMR-derived measurements of stacking equilibria, we examined the contribution of bulge-length dependent (n=0-7) stacking energetics on the binding affinity of the transactivation response element (TAR) to the arginine-rich motif Tat peptide. For most bulge lengths examined (n=2-7), we observe a striking linear relationship between the differences in stacking energetics and differences in binding energetics. This relationship was dependent on the ability to form a base triple. Interestingly, similar trends for bulge-dependent transactivation were observed in cell-based transactivation assays, though some differences were also observed that likely reflect additional interactions with the super elongation complex. Our results provide a framework for linking RNA dynamics to binding energetics in vitro and in vivo and highlight the importance of stacking energetics and base triple formation in the conformational adaptation of HIV-1 TAR.