Productive transcriptional elongation of many cellular and viral mRNAs requires transcriptional factors to extract pTEFb from the 7SK snRNP by modulating the association between the HEXIM protein and the 7SK snRNA.  Here we report the structure of the HEXIM arginine-rich motif in complex with the apical stemloop-1 of 7SK (7SK-SL1^apical) and detail how the HIV transcriptional regulator Tat from various subtypes overcome the structural constraints required to displace HEXIM. While most interactions between 7SK and HEXIM and Tat are similar, critical differences exist that guide function. First, the conformational plasticity of 7SK enables the formation of three different base pair configurations at a critical remodeling site, which allows for the modulation required for HEXIM binding and its subsequent displacement by Tat. Furthermore, the specific sequence variations observed in various Tat subtypes all converge on remodeling 7SK at this region. Second, we show that HEXIM primes its own displacement by causing specific local destabilization upon binding — a feature that is then exploited by Tat to bind 7SK more efficiently. Overall, our study details the molecular environment presented by HEXIM and uncovers a destabilization-driven displacement strategy that increases the conformational sampling of 7SK-snRNP, which may allow diverse transcriptional factors to competitively regulate pTEFb.

Isothermal titration calorimetry: Binding constants for the interactions of 7SK-SL1^apical-AGU with the HEXIM^N-ARM and Tat^Fin and Tat^G ARMs and full-length HEXIM₁ with 7SK-SL1^apical-AGU, 7SK-SL1^Full-WT, and 7SK-SL1^Full-AGU were measured using an ITC-200 microcalorimeter (MicroCal). 68 μM HEXIMN-ARM peptide was titrated into 5 μM solutions of 7SK-SL1^apical-AGU or 7SK-SL1^apicalΔASM in 10 mM sodium phosphate, 70 mM NaCl, 0.1mM EDTA, pH 5.2 at 25 °C. Titrations of Tat ARMs into 7SK-SL1^apical-AGU or 7SK-SL1^apicalΔASM were also performed in the same buffer conditions as the HEXIMN-ARM titration although the Tat ARM concentration was at 2.5 μM and the 7SK-SL1^apical-AGU concentration was at 45 μM. Titrations with full-length HEXIM1 were done at 100 μM of HEXIM1 into 3 μM of either 7SK-SL1^apical-AGU, 7SK-SL1^Full-WT, and 7SK-SL1^apical-AGU in a buffer of 25mM HEPES pH 7.5, 200mM NaCl, 5% glycerol, and 1mM TCEP. Titration curves were analyzed using ORIGIN (OriginLab) and all thermodynamic parameters are reported with n=3 experiments.

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