Monitoring tat peptide binding to TAR RNA by solid-state 31P-19F REDOR NMR

Abstract

Complexes of the HIV transactivation response element (TAR) RNA with the viral regulatory protein tat are of special interest due in particular to the plasticity of the RNA at this binding site and to the potential for therapeutic targeting of the interaction. We performed REDOR solid-state NMR experiments on lyophilized samples of a 29 nt HIV-1 TAR construct to measure conformational changes in the tat-binding site concomitant with binding of a short peptide comprising the residues of the tat basic binding domain. Peptide binding was observed to produce a nearly 4 Å decrease in the separation between phosphorothioate and 2′F labels incorporated at A27 in the upper helix and U23 in the bulge, respectively, consistent with distance changes observed in previous solution NMR studies, and with models showing significant rearrangement in position of bulge residue U23 in the bound-form RNA. In addition to providing long-range constraints on free TAR and the TAR–tat complex, these results suggest that in RNAs known to undergo large deformations upon ligand binding, 31P–19F REDOR measurements can also serve as an assay for complex formation in solid-state samples. To our knowledge, these experiments provide the first example of a solid-state NMR distance measurement in an RNA–peptide complex.

Intermediate Rate Atomic Trajectories of RNA by Solid-State NMR Spectroscopy

“…Previous work has demonstrated that phosphorothioate and 2′-fluorine substitutions at those positions do not perturb the structure of TAR26-28. These labels were added to allow us to verify the conformation of TAR RNA using REDOR methods28.…”

19F-Site-Specific-Labeled Nucleotides for Nucleic Acid Structural Analysis by NMR

“…Here, we describe a combination of RNA-labeling techniques as well as NMR methods and applications to a well-established model system, the human immunodeficiency virus (HIV) transactivation response element (TAR) (Fischer et al, 1996;Fischer, Gdaniec, Olejniczak, Bielecki, & Adamiak, 1999;Hennig et al, 2006Hennig et al, , 2007Huang, Varani, & Drobny, 2010; Olejniczak et al, 2002;Olsen et al, 2005;Scott et al, 2004). The Tat (transcription activator)-TAR complex formation provides an essential gene regulatory function for HIV (Marciniak, Calnan, Frankel, & Sharp, 1990;Seelamgari et al, 2004).…”

19F-labeling of the adenine H2-site to study large RNAs by NMR spectroscopy

“…The labeling did not affect the RNA structure or function and allowed the determination of the valine-tRNA recognition sites of the cognate aminoacyl-tRNA synthetase by 19 F-NMR spectroscopy. Since then, 2 0 F-labeled nucleotides have become widely used in solid-state and liquid-state NMR spectroscopy (Kreutz et al 2005;Olsen et al 2005). The modification on the ribosyl moiety of nucleotides allows the use of one similar labeling schema for all nucleobases.…”