The 5'-untranslated regions of all gammaretroviruses contain a conserved "double-hairpin motif" (ΨCD) 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 ([ΨCD]2, 132 nt, 42.8 kDa) using a 2H-edited NMR-spectroscopy-based approach. This approach enabled the detection of 1H-1H dipolar interactions that were not observed in previous studies of isolated SL-C and SL-D hairpin RNAs using traditional 1H-1H correlated and 1H-13C-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 [ΨCD]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, [ΨCD]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.
|Number of pages||22|
|Journal||Journal of Molecular Biology|
|State||Published - Dec 17 2010|
Bibliographical noteFunding Information:
Support from the National Institutes of Health (NIH) ( GM42561 to M.F. Summers, CA069300 to A.T., and P41RR02250 to W.C.) is gratefully acknowledged. A.S.-M. was supported by NIH MARC U⁎Star ( GM08663 ) and Howard Hughes Medical Institute education grants, and R.I. was supported by NIH Training Grant No. 5T90DK070121-05 through the Gulf Coast Consortia. We are grateful to Alexander Grishaev (National Institute of Allergy and Infectious Diseases, NIH) for collecting SAXS data and assisting in its interpretation.
- Cryo-electron tomography
- Genome packaging
- NMR spectroscopy
- RNA structure
ASJC Scopus subject areas
- Structural Biology
- Molecular Biology