Figure 1.

Secondary structure of the TAR RNA domain and variations in chemical shift and RDCs as a function of [Mg]:[TAR] stoichiometry. (a) The HIV-1 TAR analog used in this study. The six-residue hairpin loop in wt-TAR (CUGGGA) has been replaced with the more stable tetraloop (UUCG). Yellow boxes indicate residues having the largest Mg²⁺-induced ¹H chemical shift perturbations[(Δδ (ppm) = δ_TAR(Mg) – δ_TAR(free)) > 0.1 ppm, positive values imply downfield shifts]. Perturbations in ¹H chemical shifts for (b) residues in stem I (red) and II (blue) and, (c) bulge residues. RDC values measured between directly bonding C–H nuclei in (d) base moieties of residues in stems I (red) and II (blue) and (e) base and sugar moieties of bulge residues. Uniformly labeled ¹⁵N/¹³C-labeled TAR was prepared using standard procedures as described.²¹ NMR samples contained ~1.0 mM ¹⁵N/¹³C-labeled TAR, 15 mM sodium phosphate (pH 6.0–6.2), 25 mM sodium sulfate, 0.1 mM EDTA, and 3.0 and 4.5 mM Mg²⁺ for TAR(3.0 Mg) and TAR(4.5 Mg), respectively. An identical NMR sample also contained ~21 mg/ml of Pf1 phage.^34,35 All NMR data were acquired on Varian Inova spectrometers operating at ¹H frequencies of 500 and 600 MHz at 25 °C. Resonances were assigned by following incremental shifts upon addition of Mg²⁺. As described,²¹ splittings between [C8–H8, C6–H6, C2–H2], [C5–H5], [C1′ –H1′] were measured using ¹H and ¹³C IPAP-HSQC experiments⁴⁰ and between [C2′ –H2′, C3′ –H3′, C4′ –H4′] using the ¹J_CH-CT-CE-HSQC experiment.¹⁶ RDCs were calculated as the differences between splittings measured in the presence and absence of phage. The uncertainty in RDCs, estimated from measuring splittings in the ¹H and ¹³C dimension, was <3.5 Hz and <4.5 Hz for TAR(3.0 Mg) and TAR(4.5 Mg), respectively. The greater uncertainty in RDCs measured in the presence of Mg²⁺ relative to TAR(free)²¹ is attributed to increased line widths arising from greater degree of alignment as well as exchange broadening due to non-specific Mg²⁺ binding.