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Book of Abstracts: Albany 2007

category image Albany 2007
Conversation 15
June 19-23 2007

Nucleic Acid Chaperone Activity of Retroviral Nucleocapsid Proteins

The nucleocapsid proteins (NC) of different retroviruses are known to possess the nucleic acid (NA) chaperone activity, i.e. the ability to facilitate kinetics of NA molecules restructuring to their lowest energy state. Previous research suggested that the ability of NC proteins to destabilize secondary NA structures, aggregate NA and have a fast NA/protein interaction kinetics to be major contributors to the NA chaperone activity. In this work we use gel-shift annealing and sedimentation assays, as well as single molecule DNA stretching in the presence of NC proteins to quantitatively characterize all of these aspects of NC chaperone function. We follow the annealing of the complementary DNA and RNA hairpins derived from the HIV genome that mimic the annealing step of the minus-strand transfer reaction in reverse transcription. Hairpin annealing kinetics is studied over a wide range of HIV-1 NC, Na+ and Mg2+ cations and DNA concentrations and temperature. In addition, we studied various NC protein mutants and truncated versions to determine contribution of different NC structural features to its chaperone function. We also studied NC proteins from several retroviruses, such as HIV-1, MLV, RSV and HTLV-1. We find that the major contribution to NC?s chaperon function comes from its ability to aggregate NA similarly to the simple multivalent cation with the effective +3 charge, thereby facilitating the bimolecular nucleation step of annealing by 102-105 fold, dependent on solution conditions. The smaller (less then 10-fold) facilitation comes from the NC?s.

Ioulia Rouzina1
My-Nuong Vo1
Kristen Stewart Maynard1
Margareta Cruceanu2
Mark Williams2
Karin Musier-Forsyth1

1University of Minnesota, Department of Chemistry and Department of Molecular Biology, Biochemistry and Biophysics, Minneapolis MN, 1
Northesatern University, Boston, MA.

Tel: 612-624-7468
Fax: 612-624-5121
rouzi002@umn.edu