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Follow the RNA leader

SBKB [doi:10.1038/sbkb.2011.53]
Technical Highlight - December 2011
Short description: An NMR method characterizes an RNA structural switch that regulates HIV-1 genome packaging.

Residues near the gag start codon (AUG, green) form a hairpin in the monomeric RNA, and residues that promote dimerization (DIS, red) are sequestered by base-pairing with U5. Base-pairing between AUG and U5 displaces and exposes the DIS, which enables dimerization. Image courtesy of Michael F. Summers.

Like all retroviruses, HIV-1 packages a dimeric copy of its RNA genome during replication. The highly conserved 5′-leader region of the genome facilitates this process via interactions with the nucleocapsid (NC) domain of the viral Gag polyprotein. Although structures of the constituent RNA stem-loops are known, a consensus on the global structure of the 5′-leader has not emerged. However, there is evidence that both dimerization and subsequent packaging are mechanistically coupled to translational control and require the essential AUG region containing the gag start codon. A key question lies in understanding if the 5′-leader maintains a structural equilibrium dependent on whether the genome is undergoing active translation or has switched to dimerization and packaging.

Summers, Telesnitsky and colleagues have developed an innovative NMR method to study the structures adopted by the 5′-leader. Initial experiments allowed the authors to define buffer conditions favoring either monomer (115 kDa) or dimer (230 kDa) formation and quantify the equilibrium governing the conformational transition. Next, enzymatic ligation of an NMR-active 13C-enriched AUG region in an otherwise unlabeled background allowed them to selectively observe NMR signals from this critical sequence in the intact 5′-leader RNA.

The authors then sought to resolve conflicting models for the interaction of the AUG region with other regions of the RNA in both the monomeric and dimeric forms. By designing RNA containing a unique [UUA:UAA] element that has distinctive adenosine NMR signals, they found that the AUG region is in a relatively mobile stem-loop in the monomer but base-pairs with the upstream unique-5′ element (U5) in the dimer. This structural rearrangement sequesters the gag start codon and disrupts interactions between the U5 element and the dimerization site. High-affinity binding sites for NC are then exposed in the dimer as confirmed by isothermal titration calorimetry experiments and increased packaging during in vivo assays. The combination of enzymatic ligation and conservative mutagenesis therefore allowed the authors to use simple NMR experiments to characterize an RNA conformational transition that regulates viral replication. This method extends the range of mechanisms that can be probed in large RNA systems.

Michael A. Durney

References

  1. K. Lu et al. NMR detection of structures in the HIV-1 5′-leader RNA that regulate genome packaging.
    Science 334, 242-245 (2011). doi:10.1126/science.1210460

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Funded by a grant from the National Institute of General Medical Sciences of the National Institutes of Health