Robert D. Peterson

Correlation of nucleotide base and sugar protons in a 15N-labeled HIV-1 RNA oligonucleotide by 1H-15N HSQC experiments.

SummaryThe advent of methods for preparing 15N- and 13C-labeled RNA oligonucleotides holds promise for extending the size of RNA molecules that can be studies by NMR spectroscopy. A practical limitation is the expense of the 13C label. It may therefore sometimes be desirable to prepare a relatively inexpensive 15N-labeled sample only. Here we show that the two-bond 1H-15N HSQC experiment can be used on 15N-labeled RNA to correlate the intranucleotide H1′ and H8,H6,H5 resonances indirectly through the shared glycosidic nitrogen. The nonrefocused version of a standard HSQC experiment for 2D proton-detected 1H-15N chemical-shift correlation is applied in order to minimize the sensitivity loss due to the relatively fast spin-spin relaxation of RNA oligonucleotides. The experiment is applied to the 30-nucleotide RNA RBE3 which contains the high-affinity binding site of the RRE (rev response element) for the Rev protein of HIV. The results indicate that this simple experiment allows a straightforward identification of the base proton resonances CH5, CH6, UH5, UH6, purine H8, and AH2 as well as the intranucleotide H1′ and H8,H6,H5 connectivities. When combined with a NOESY experiment, complete sequential assignments can be obtained.

Structurally conserved five nucleotide bulge determines the overall topology of the core domain of human telomerase RNA

Telomerase is a unique ribonucleoprotein complex that catalyzes the addition of telomeric DNA repeats onto the 3′ ends of linear chromosomes. All vertebrate telomerase RNAs contain a catalytically essential core domain that includes the template and a pseudoknot with extended helical subdomains. Within these helical regions is an asymmetric 5-nt internal bulge loop (J2a/b) flanked by helices (P2a and P2b) that is highly conserved in its location but not sequence. NMR structure determination reveals that J2a/b forms a defined S-shape and creates an ∼90 ° bend with a surprisingly low twist (∼10 °) between the flanking helices. A search of RNA structures revealed only one other example of a 5-nt bulge, from hepatitis C virus internal ribosome entry site, with a different sequence but the same structure. J2a/b is intrinsically flexible but the interhelical motions across the loop are remarkably restricted. Nucleotide substitutions in J2a/b that affect the bend angle, direction, and interhelical dynamics are correlated with telomerase activity. Based on the structures of P2ab (J2a/b and flanking helices), the conserved region of the pseudoknot (P2b/P3, previously determined) and the remaining helical segment (P2a.1–J2a.1 refined using residual dipolar couplings and the modeling program MC-Sym) we have calculated an NMR-based model of the full-length pseudoknot. The model and dynamics analysis show that J2a/b serves as a dominant structural and dynamical element in defining the overall topology of the core domain, and suggest that interhelical motions in P2ab facilitate nucleotide addition along the template and template translocation.

Comparison of solution and crystal structures of preQ1 riboswitch reveals calcium-induced changes in conformation and dynamics.

Riboswitches regulate gene expression via specific recognition of cognate metabolites by their aptamer domains, which fold into stable conformations upon ligand binding. However, the recently reported solution and crystal structures of the Bacillus subtilis preQ1 riboswitch aptamer show small but significant differences, suggesting that there may be conformational heterogeneity in the ligand-bound state. We present a structural and dynamic characterization of this aptamer by solution NMR spectroscopy. The aptamer−preQ1 complex is intrinsically flexible in solution, with two regions that undergo motions on different time scales. Three residues move in concert on the micro-to-millisecond time scale and may serve as the lid of the preQ1-binding pocket. Several Ca2+ ions are present in the crystal structure, one of which binds with an affinity of 47 ± 2 μM in solution to a site that is formed only upon ligand binding. Addition of Ca2+ to the aptamer−preQ1 complex in solution results in conformational changes that account for the differences between the solution and crystal structures. Remarkably, the Ca2+ ions present in the crystal structure, which were proposed to be important for folding and ligand recognition, are not required for either in solution.

A Heteronuclear Zero Quantum Coherence Nz-Exchange Experiment That Resolves Resonance Overlap and Its Application To Measure the Rates of Heme Binding to the IsdC Protein

Chemical exchange phenomena in NMR spectra can be quantitatively interpreted to measure the rates of ligand binding, as well as conformational and chemical rearrangements. In macromolecules, processes that occur slowly on the chemical shift time scale are frequently studied using 2D heteronuclear ZZ or N(z)-exchange spectroscopy. However, to successfully apply this method, peaks arising from each exchanging species must have unique chemical shifts in both dimensions, a condition that is often not satisfied in protein-ligand binding equilibria for (15)N nuclei. To overcome the problem of (15)N chemical shift degeneracy we developed a heteronuclear zero-quantum (and double-quantum) coherence N(z)-exchange experiment that resolves (15)N chemical shift degeneracy in the indirect dimension. We demonstrate the utility of this new experiment by measuring the heme binding kinetics of the IsdC protein from Staphylococcus aureus. Because of peak overlap, we could not reliably analyze binding kinetics using conventional methods. However, our new experiment resulted in six well-resolved systems that yielded interpretable data. We measured a relatively slow k(off) rate of heme from IsdC (<10 s(-1)), which we interpret as necessary so heme loaded IsdC has time to encounter downstream binding partners to which it passes the heme. The utility of using this new exchange experiment can be easily expanded to (13)C nuclei. We expect our heteronuclear zero-quantum coherence N(z)-exchange experiment will expand the usefulness of exchange spectroscopy to slow chemical exchange events that involve ligand binding.

Compensating increases in protein backbone flexibility occur when the Dead ringer AT-rich interaction domain (ARID) binds DNA: a nitrogen-15 relaxation study.

AT‐rich interaction domains (ARIDs) are found in a large number of eukaryotic transcription factors that regulate cell proliferation, differentiation, and development. Previously we elucidated how ARIDs recognize DNA by determining the solution structure of the Drosophila melanogaster Dead ringer protein in both its DNA‐free and ‐bound states. In order to quantitatively determine how ARIDs alter their mobility to recognize DNA, we have measured the relaxation parameters of the backbone nitrogen‐15 nuclei of Dead ringer in its free and bound forms, and interpreted these data using the model‐free approach. We show that Dead ringer undergoes significant changes in its mobility upon binding, with residues in the loop connecting helices H5 and H6 becoming immobilized in the major groove and contacts to the minor groove slowing down the motion of residues at the C terminus. A DNA‐induced rotation and displacement of the N‐terminal subdomain of the protein increases the mobility of helix H1 located distal to the DNA interface and may partially negate the entropic cost of immobilizing interfacial residues. Elevated motions on the micro‐ to millisecond timescale in the N‐terminal domain prior to DNA binding appear to foreshadow the DNA‐induced conformation change.