Journal
EUROPEAN JOURNAL OF MASS SPECTROMETRY
Volume 13, Issue 1, Pages 29-33Publisher
SAGE PUBLICATIONS LTD
DOI: 10.1255/ejms.839
Keywords
hIV-1; RNA isomerization; RNA conformation; ribonucleoprotein; protein-nucleic acids assembly; non-covalent complex; protein binding; chaperone; competitive binding; ESI-FT-ICR
Funding
- NIGMS NIH HHS [R01-GM643208] Funding Source: Medline
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Nanospray-FT-ICR has been employed to investigate the processes of genome dimerization, selection, and packaging in human immunodifficiency virus type 1, which are mediated by specific interactions between the nucleocapsid protein (NQ and the structural elements formed by the genome's packaging signal (Psi-ribonucleic acid (RNA)]. This analytical platform allowed for the unambiguous characterization of all the non-covalent complexes formed in vitro by simultaneous RNA-RNA and protein-RNA binding equilibria. Competitive binding experiments involving the isolated RNA elements were completed to evaluate their ability to sustain specific protein interactions. In similar fashion, ad hoc RNA mutants were used to locate two distinct binding sites on the apical loop and stem-bulge of the monomeric stemloop 1 (SL1) domain, which is responsible for initiating the dimerization process. The stem-bulge motifs provided viable binding sites in both the kissing-loop (KL) and the extended duplex forms of dimeric SL1, whereas the latter included additional sites corresponding to the A-bulge motifs that flank the annealed palindromes. A cross-linking approach using pre-derivatized, photo - cross-linkable NC demonstrated that the SL3 domain was the preferred site for protein binding in the context of full-length Psi-RNA. This concerted strategy is expected to provide new valuable insight into the effects induced by the global folding of Psi-RNA on its ability to interact with the NC protein during genome dimerization, selection and packaging.
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