Nucleic Acids Research Advance Access originally published online on March 6, 2007
Nucleic Acids Research 2007 35(6):2026-2034; doi:10.1093/nar/gkm097
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Nucleic Acids Research, 2007, Vol. 35, No. 6 2026-2034
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Structural Biology |
Nucleocapsid protein-mediated maturation of dimer initiation complex of full-length SL1 stemloop of HIV-1: sequence effects and mechanism of RNA refolding
1Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, CA 94158-2517, USA and 2Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA
*To whom correspondence should be addressed. Tel: +1-415 476-1916; Fax: +1-415 502 8298; Email: james{at}picasso.ucsf.edu
Received December 15, 2006. Revised February 2, 2007. Accepted February 1, 2007.
Specific binding of HIV-1 viral protein NCp7 to a unique 35-base RNA stem-loop SL1 is critical for formation and packaging of the genomic RNA dimer found within HIV-1 virions. NCp7 binding stimulates refolding of SL1 from a metastable kissing dimer (KD) into thermodynamically stable linear dimer (LD). Using UV melting, gel electrophoresis and heteronuclear NMR, we investigated effects of various site-specific mutations within the full-length SL1 on temperature- or NCp7-induced refolding in vitro. Refolding involved intramolecular melting of SL1 stems but not dissociation of the intermolecular KD interface. Refolding required only two NCp7 molecules per KD but was limited by the amount of NCp7 present, implying that the protein does not catalytically promote refolding. Efficient refolding depended strictly on the presence and, to a lesser degree, on sequence of a highly conserved G-rich internal loop that normally limits thermal stability of the SL1 stem. Adding two base pairs to the lower stem created a hyperstable SL1 mutant that failed to refold, even when bound by NCp7 at high stoichiometries. NMR analysis of these kinetically trapped mutant RNA–protein complexes indicated that NCp7 initiates refolding by dissociating base pairs in the upper stem of SL1. This study illuminates structural transitions critical for HIV-1 assembly and replication.
Present address: Anwer Mujeeb, Universitywide AIDS Research Program, University of California, Oakland, CA 94612-3550, USA
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  N. B. Ulyanov, K. Shefer, T. L. James, and Y. Tzfati Pseudoknot structures with conserved base triples in telomerase RNAs of ciliates Nucleic Acids Res., September 25, 2007; 35(18): 6150 - 6160. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. T. Baig, J.-M. Lanchy, and J. S. Lodmell HIV-2 RNA dimerization is regulated by intramolecular interactions in vitro RNA, August 1, 2007; 13(8): 1341 - 1354. [Abstract] [Full Text] [PDF]
|
|