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Understanding the role of bulge‐length dependent RNA stacking energetics in determining HIV‐1 TAR‐Tat peptide binding energetics in vitro and in cells
Author(s) -
Kelly Megan,
Roy Rohit,
Bogerd Hal,
Merriman Dawn,
Ganser Laura,
Cullen Bryan,
AlHashimi Hashim
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2021.35.s1.02346
Subject(s) - energetics , stacking , biophysics , chemistry , rna , transactivation , binding site , molecular dynamics , plasma protein binding , crystallography , biology , biochemistry , computational chemistry , transcription factor , ecology , organic chemistry , gene
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.