Lucy Fallon, PhD
New York University
A Shifting Landscape: pH and Mg2+ Modulate the Conformational Landscape of 7SK SL-I Through Orthogonal Mechanisms
The small-nuclear RNA (snRNA) 7SK is essential for regulating the transcription of genes by RNA polymerase II through its interactions with Hexamethylene Bisacetamide Inducible Protein 1 (HEXIM) and Positive Transcription Elongation Factor b (P-TEFb). High-resolution structures of 7SK stem-loop 1 (SL-1) reveal the presence of several base triples that coordinate the interaction with HEXIM. However, different high-resolution structures of 7SK SL-1 exhibit different conformations, each with unique base triple configurations. Previous NMR studies show that the conformation of 7SK SL-1 is sensitive to changes in pH and magnesium (Mg2+) concentration. However, the exact mechanism by which Mg2+ and pH influence the formation and stability of base triples in 7SK SL-1 at an atomic level remains unclear.
We combine molecular dynamics simulations, free energy calculations, nuclear magnetic resonance, and small angle X-ray scattering to characterize the mechanism and dynamics of base triple formation in 7SK SL-1, and deconvolute the effects of pH and Mg2+. We find that the conformational landscape of 7SK SL-1 dramatically changes depending on the pH and presence of Mg2+, and that pH and Mg2+ act in orthogonal ways to favor the formation of base triples. We discuss how the dynamic behavior of the base triples may provide a means for 7SK to regulate its interactions with HEXIM, modulating the transcriptional output of RNA polymerase II.
The small-nuclear RNA (snRNA) 7SK is essential for regulating the transcription of genes by RNA polymerase II through its interactions with Hexamethylene Bisacetamide Inducible Protein 1 (HEXIM) and Positive Transcription Elongation Factor b (P-TEFb). High-resolution structures of 7SK stem-loop 1 (SL-1) reveal the presence of several base triples that coordinate the interaction with HEXIM. However, different high-resolution structures of 7SK SL-1 exhibit different conformations, each with unique base triple configurations. Previous NMR studies show that the conformation of 7SK SL-1 is sensitive to changes in pH and magnesium (Mg2+) concentration. However, the exact mechanism by which Mg2+ and pH influence the formation and stability of base triples in 7SK SL-1 at an atomic level remains unclear.
We combine molecular dynamics simulations, free energy calculations, nuclear magnetic resonance, and small angle X-ray scattering to characterize the mechanism and dynamics of base triple formation in 7SK SL-1, and deconvolute the effects of pH and Mg2+. We find that the conformational landscape of 7SK SL-1 dramatically changes depending on the pH and presence of Mg2+, and that pH and Mg2+ act in orthogonal ways to favor the formation of base triples. We discuss how the dynamic behavior of the base triples may provide a means for 7SK to regulate its interactions with HEXIM, modulating the transcriptional output of RNA polymerase II.