Characterizing the relative orientation and dynamics of RNA A-form helices using NMR residual dipolar couplings - PubMed (original) (raw)

Characterizing the relative orientation and dynamics of RNA A-form helices using NMR residual dipolar couplings

Maximillian H Bailor et al. Nat Protoc. 2007.

Abstract

We present a protocol for determining the relative orientation and dynamics of A-form helices in 13C/15N isotopically enriched RNA samples using NMR residual dipolar couplings (RDCs). Non-terminal Watson-Crick base pairs in helical stems are experimentally identified using NOE and trans-hydrogen bond connectivity and modeled using the idealized A-form helix geometry. RDCs measured in the partially aligned RNA are used to compute order tensors describing average alignment of each helix relative to the applied magnetic field. The order tensors are translated into Euler angles defining the average relative orientation of helices and order parameters describing the amplitude and asymmetry of interhelix motions. The protocol does not require complete resonance assignments and therefore can be implemented rapidly to RNAs much larger than those for which complete high-resolution NMR structure determination is feasible. The protocol is particularly valuable for exploring adaptive changes in RNA conformation that occur in response to biologically relevant signals. Following resonance assignments, the procedure is expected to take no more than 2 weeks of acquisition and data analysis time.

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Figures

Figure 1

RDCs (Dij) between spins i and j provide long-range constraints on the average orientation (θ) of the internuclear bond vector relative to the applied magnetic field.

Figure 2

Determining the relative orientation and dynamics of A-form helices using an order tensor analysis of RDCs, . (a) Watson-Crick (WC) base pairs that are flanked by other WC base pairs are identified based on the predicted RNA secondary structural model. Resonance assignments in WC base pairs are established using NOESY connectivity (shown in the figure) or using through bond correlation experiments. The local structure of the experimentally verified WC pairs is modeled using the idealized A-form geometry. Next, NMR experiments (Table 1) are used to measure splittings between various nuclei under aligned (J + D) (Table 2) and unaligned (J) conditions. Note that differences in the chemical shifts (center of doublet) between aligned and unaligned conditions arise owing to a combination of RCSA contributions and different lock frequencies as a result of quadrupolar splitting of the D2O signal in the aligned state. RDCs are computed from the differences in these values and, together with the idealized A-form PDBs, are used to compute order tensors for each helix. (b) The helix order tensor frames (Szz, Syy, Sxx) are superimposed to yield the relative orientation of helices subject to a 4_n_−1-fold degeneracy arising owing to allowed 180° inversions about the principal Szz, Syy and Sxx directions. Information about interhelix motions is obtained from the relative ratios of the generalized degree of order (ϑ) obtained for each helix (ϑ_i_ /ϑ_j_ = ϑint; ϑ_i_ <ϑ_j_). The ϑint value ranges between 0 for maximum interhelix motions and 1 for interhelix rigidity. Owing to motional couplings, the ϑint value will generally underestimate the real amplitude of interhelical motions.

Figure 3

Typical RDCs measured in base and sugar moieties of RNA using the pulse sequences listed in Table 1. (a) One-bond C–H and N–H RDCs are the most commonly targeted interactions owing to their favorable size but smaller one-bond C–C and C–N as well as (b) two- and three-bond RDCs can be measured. The motionally non-averaged C–H and N–H bond lengths used in the order tensor analysis are N1/3–H1/3 = 1.01 Å, C–Hbase = 1.08 Å, C–Hribose = 1.09 Å (ref. 100). All other bond lengths can be obtained from ref. .

Figure 4

Example illustrating application of protocol in the determination of the relative orientation and dynamics of two helices in the free state of HIV-1 TAR RNA. (a) Idealized A-form helices are used to determine order tensors for each helix in TAR RNA, with 18 (12 base, 6 sugar) and 22 (13 base, 9 sugar) one-bond C–H RDCs used in the analysis of helices I and II, respectively. (b) Superposition of the experimentally determined order tensor frames yields one of four solutions for the relative orientation of helices. Three additional degenerate solutions (180° Szz, 180° Syy, 180° Sxx) are generated by subsequent rotation of a given helix (in this case helix II) by 180° about each of the three helix II principal axes Szz, Syy and Sxx, respectively. In each case, the helices are translated/assembled by setting the distance between 03′ of residue 39 and P of residue 40 equal to 1.58 Å. The 180° Szz solution is discarded because it yields a distance between 03′ of residue 22 and P of residue 40 that is long to be satisfactorily connected by a trinucleotide bulge, whereas solutions 180° Syy and 180° Sxx are discarded because they lead to an antiparallel helix alignment that is inconsistent with the TAR secondary structure.

Figure 5

The relative orientation (interhelix bend, θh, and twist angles, ξ) and dynamics (ϑint) of RNA helices obtained from order tensor analysis of RDCs under different contexts. (a) Three different RNA secondary structures (TAR, SL1, P4) in the presence and absence of Mg2+ (see refs. 21,22,87). (b) TAR RNA in four distinct ligand-bound forms,,. The ϑint values are color-coded on each point. The large uncertainty in the interhelix twist angles (set at ± 50° (see ref. 102)) arises from near axial symmetry (η~0) of the order tensor. The net charge delivered by each small molecule upon binding is shown in parentheses. Although the ligand arginanamide (Arg) has a +2 charge, studies show that up to three molecules can bind to TAR contributing a total +6 charge. For Mg2+, the charge of +8 is shown based on the observation of four bound Ca2+ ions in the X-ray structure of TAR.

References

1. Williamson JR. Molecular biology—small subunit, big science. Nature. 2000;407:306–307. - PubMed
1. Leulliot N, Varani G. Current topics in RNA-protein recognition: control of specificity and biological function through induced fit and conformational capture. Biochemistry. 2001;40:7947–7956. - PubMed
1. Al-Hashimi HM. Dynamics-based amplification of RNA function and its characterizationbyusing NMR spectroscopy. ChemBioChem. 2005;6:1506–1519. - PubMed
1. Micura R, Hobartner C. On secondary structure rearrangements and equilibria of small RNAs. ChemBioChem. 2003;4:984–990. - PubMed
1. Williamson JR. Assembly of the 30S ribosomal subunit. Q. Rev. Biophys. 2005;38:397–403. - PubMed

Characterizing the relative orientation and dynamics of RNA A-form helices using NMR residual dipolar couplings - PubMed (original) (raw)