Journal: J Mol Biol / Year: 2010 Title: Structure of a conserved retroviral RNA packaging element by NMR spectroscopy and cryo-electron tomography. Authors: Yasuyuki Miyazaki / Rossitza N Irobalieva / Blanton S Tolbert / Adjoa Smalls-Mantey / Kilali Iyalla / Kelsey Loeliger / Victoria D'Souza / Htet Khant / Michael F Schmid / Eric L Garcia / ...Authors: Yasuyuki Miyazaki / Rossitza N Irobalieva / Blanton S Tolbert / Adjoa Smalls-Mantey / Kilali Iyalla / Kelsey Loeliger / Victoria D'Souza / Htet Khant / Michael F Schmid / Eric L Garcia / Alice Telesnitsky / Wah Chiu / Michael F Summers / Abstract: The 5'-untranslated regions of all gammaretroviruses contain a conserved "double-hairpin motif" (Ψ(CD)) that is required for genome packaging. Both hairpins (SL-C and SL-D) contain GACG tetraloops ...The 5'-untranslated regions of all gammaretroviruses contain a conserved "double-hairpin motif" (Ψ(CD)) that is required for genome packaging. Both hairpins (SL-C and SL-D) contain GACG tetraloops that, in isolated RNAs, are capable of forming "kissing" interactions stabilized by two intermolecular G-C base pairs. We have determined the three-dimensional structure of the double hairpin from the Moloney murine leukemia virus ([Ψ(CD)](2), 132 nt, 42.8 kDa) using a (2)H-edited NMR-spectroscopy-based approach. This approach enabled the detection of (1)H-(1)H dipolar interactions that were not observed in previous studies of isolated SL-C and SL-D hairpin RNAs using traditional (1)H-(1)H correlated and (1)H-(13)C-edited NMR methods. The hairpins participate in intermolecular cross-kissing interactions (SL-C to SL-D' and SLC' to SL-D) and stack in an end-to-end manner (SL-C to SL-D and SL-C' to SL-D') that gives rise to an elongated overall shape (ca 95 Å×45 Å×25 Å). The global structure was confirmed by cryo-electron tomography (cryo-ET), making [Ψ(CD)](2) simultaneously the smallest RNA to be structurally characterized to date by cryo-ET and among the largest to be determined by NMR. Our findings suggest that, in addition to promoting dimerization, [Ψ(CD)](2) functions as a scaffold that helps initiate virus assembly by exposing a cluster of conserved UCUG elements for binding to the cognate nucleocapsid domains of assembling viral Gag proteins.
Method: distance geometry / Software ordinal: 1 Details: Structures were calculated and refined with CYANA using the AMBER residue library. Upper-limit distance restraints of 2.7 , 3.3 and 5.0 were employed for direct NOE cross peaks of strong, ...Details: Structures were calculated and refined with CYANA using the AMBER residue library. Upper-limit distance restraints of 2.7 , 3.3 and 5.0 were employed for direct NOE cross peaks of strong, medium and weak intensities, respectively, for all cross peaks except those associated with the intraresidue H8/6-to-H2 and H3 interactions. For these proton pairs, upper distance limits of 4.2 and 3.2 were therefore employed for NOEs of medium and strong intensity, respectively.43 Cross-helix P-P distance restraints (with 20% weighting coefficient) were employed for A-form helical segments to prevent the generation of structures with collapsed major grooves:43,52,55 P(i)-P(i+2) (cross-helix phosphorus of the i+2 base pair) = 16.1 - 17.1 , P(i)-P(i+3) = 14.2 - 15.2 ; P(i)-P(i+4) = 11.7 -12.7 ; P(i)-P(i+5) = 9.4 -10.4 ; P(i)-P(i+6) = 9.0 -10.0 . Torsion angle restraints for A helical stem residues were centered around published A-form RNA values 112 with allowed deviations of 50 degrees. Four restraints per hydrogen bond were employed to enforce approximately linear NH-N and NH-O bond distances of 1.85 0.05, and two lower limit restraints per base pair were employed to weakly enforce base pair planarity (20% weighting coefficient) (G-C base pairs: G-C4 to C-C6 > 8.3 and G-N9 to C-H6 > 10.75 . A-U base pairs: A-C4 to U-C6 > 8.3 and A-N9 to U-H6 > 10.75 ).
NMR representative
Selection criteria: lowest energy
NMR ensemble
Conformer selection criteria: structures with the lowest energy Conformers calculated total number: 340 / Conformers submitted total number: 20
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