Major groove width variations in RNA structures determined by NMR and impact of 13C residual chemical shift anisotropy and 1H-13C residual dipolar coupling on refinement.

Ribonucleic acid structure determination by NMR spectroscopy relies primarily on local structural restraints provided by (1)H- (1)H NOEs and J-couplings. When employed loosely, these restraints are broadly compatible with A- and B-like helical geometries and give rise to calculated structures that are highly sensitive to the force fields employed during refinement. A survey of recently reported NMR structures reveals significant variations in helical parameters, particularly the major groove width. Although helical parameters observed in high-resolution X-ray crystal structures of isolated A-form RNA helices are sensitive to crystal packing effects, variations among the published X-ray structures are significantly smaller than those observed in NMR structures. Here we show that restraints derived from aromatic (1)H- (13)C residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs) can overcome NMR restraint and force field deficiencies and afford structures with helical properties similar to those observed in high-resolution X-ray structures.

An RNA structural switch regulates diploid genome packaging by Moloney murine leukemia virus.

Retroviruses selectively package two copies of their RNA genomes via mechanisms that have yet to be fully deciphered. Recent studies with small fragments of the Moloney murine leukemia virus (MoMuLV) genome suggested that selection may be mediated by an RNA switch mechanism, in which conserved UCUG elements that are sequestered by base-pairing in the monomeric RNA become exposed upon dimerization to allow binding to the cognate nucleocapsid (NC) domains of the viral Gag proteins. Here we show that a large fragment of the MoMuLV 5' untranslated region that contains all residues necessary for efficient RNA packaging (Psi(WT); residues 147-623) also exhibits a dimerization-dependent affinity for NC, with the native dimer ([Psi(WT)](2)) binding 12+/-2 NC molecules with high affinity (K(d)=17+/-7 nM) and with the monomer, stabilized by substitution of dimer-promoting loop residues with hairpin-stabilizing sequences (Psi(M)), binding 1-2 NC molecules. Identical dimer-inhibiting mutations in MoMuLV-based vectors significantly inhibit genome packaging in vivo (approximately 100-fold decrease), whereas a large deletion of nearly 200 nucleotides just upstream of the gag start codon has minimal effects. Our findings support the proposed RNA switch mechanism and further suggest that virus assembly may be initiated by a complex comprising as few as 12 Gag molecules bound to a dimeric packaging signal.