Biomaterial Database

Mechanistic studies of mini-TAR RNA/DNA annealing in the absence and presence of HIV-1 nucleocapsid protein. 2006

My-Nuong Vo, and George Barany, and Ioulia Rouzina, and Karin Musier-Forsyth

University of Minnesota, Department of Chemistry and Institute for Molecular Virology, Minneapolis, MN 55455, USA.

HIV-1 reverse transcription involves several nucleic acid rearrangements, which are catalyzed by the nucleocapsid protein (NC). Annealing of the trans-activation response element (TAR) DNA hairpin to a complementary TAR RNA hairpin, resulting in the formation of an extended 98-base-pair duplex, is an essential step in the minus-strand transfer step of reverse transcription. To elucidate the TAR RNA/DNA annealing reaction pathway, annealing kinetics were studied systematically by gel-shift assays performed in the presence or absence of HIV-1 NC. Truncated 27 nucleotide mini-TAR RNA and DNA constructs were used in this work. In the absence of NC, the annealing is slow, and involves the fast formation of an unstable extended "kissing" loop intermediate, followed by a slower strand exchange between the terminal stems. This annealing is very sensitive to loop-loop complementarity, as well as to nucleic acid concentration, ionic strength and temperature. NC stimulates the annealing approximately 5000-fold by stabilizing the bimolecular intermediate approximately 100 to 200-fold, and promoting the subsequent strand exchange reaction approximately 10 to 20-fold. NC concentration dependence studies suggest that there is a direct correlation between the amount of NC required to stabilize the intermediate and the amount needed to induce mini-TAR aggregation. Whereas saturating levels of NC are required to efficiently aggregate nucleic acids, sub-saturating NC is sufficient to significantly enhance duplex destabilization. Equilibrium levels of mini-TAR RNA/DNA annealing were also measured under a variety of conditions. Taken together, the results presented here provide a quantitative accounting of HIV-1 NC's aggregation and duplex destabilizing activity, and provide insights into the universal nucleic acid chaperone activity of this essential viral protein.

UI	MeSH Term	Description	Entries
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D009690	Nucleic Acid Conformation	The spatial arrangement of the atoms of a nucleic acid or polynucleotide that results in its characteristic 3-dimensional shape.	DNA Conformation,RNA Conformation,Conformation, DNA,Conformation, Nucleic Acid,Conformation, RNA,Conformations, DNA,Conformations, Nucleic Acid,Conformations, RNA,DNA Conformations,Nucleic Acid Conformations,RNA Conformations
D009692	Nucleic Acid Heteroduplexes	Double-stranded nucleic acid molecules (DNA-DNA or DNA-RNA) which contain regions of nucleotide mismatches (non-complementary). In vivo, these heteroduplexes can result from mutation or genetic recombination; in vitro, they are formed by nucleic acid hybridization. Electron microscopic analysis of the resulting heteroduplexes facilitates the mapping of regions of base sequence homology of nucleic acids.	Heteroduplexes, Nucleic Acid,Heteroduplex DNA,Acid Heteroduplexes, Nucleic,DNA, Heteroduplex
D004279	DNA, Viral	Deoxyribonucleic acid that makes up the genetic material of viruses.	Viral DNA
D001483	Base Sequence	The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence.	DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D012367	RNA, Viral	Ribonucleic acid that makes up the genetic material of viruses.	Viral RNA
D015497	HIV-1	The type species of LENTIVIRUS and the etiologic agent of AIDS. It is characterized by its cytopathic effect and affinity for the T4-lymphocyte.	Human immunodeficiency virus 1,HIV-I,Human Immunodeficiency Virus Type 1,Immunodeficiency Virus Type 1, Human
D016325	HIV Long Terminal Repeat	Regulatory sequences important for viral replication that are located on each end of the HIV genome. The LTR includes the HIV ENHANCER, promoter, and other sequences. Specific regions in the LTR include the negative regulatory element (NRE), NF-kappa B binding sites , Sp1 binding sites, TATA BOX, and trans-acting responsive element (TAR). The binding of both cellular and viral proteins to these regions regulates HIV transcription.	HIV Negative Regulatory Element,HIV Sp1-Binding Site,HIV Trans-Acting Responsive Region,Human Immunodeficiency Virus Long Terminal Repeat,Long Terminal Repeat, HIV,Negative Regulatory Element, HIV,Sp1-Binding Site, HIV,Trans-Acting Responsive Region, HIV,HIV-1 LTR,Human Immunodeficiency Virus LTR,LTR, Human Immunodeficiency Virus,TAR Element, HIV,Trans-Activation Responsive Element, HIV,Trans-Activation Responsive Region, HIV,HIV 1 LTR,HIV Sp1 Binding Site,HIV Sp1-Binding Sites,HIV TAR Element,HIV TAR Elements,HIV Trans Acting Responsive Region,LTR, HIV-1,Sp1 Binding Site, HIV,Sp1-Binding Sites, HIV,TAR Elements, HIV,Trans Acting Responsive Region, HIV,Trans Activation Responsive Element, HIV,Trans Activation Responsive Region, HIV
D019590	Nucleocapsid Proteins	Viral proteins found in either the NUCLEOCAPSID or the viral core (VIRAL CORE PROTEINS).	Viral N Proteins,Viral Nucleoproteins,Nucleocapsid Protein,Viral N Protein,N Protein, Viral,N Proteins, Viral,Nucleoproteins, Viral,Proteins, Viral N
D020218	Response Elements	Nucleotide sequences, usually upstream, which are recognized by specific regulatory transcription factors, thereby causing gene response to various regulatory agents. These elements may be found in both promoter and enhancer regions.	Element, Response,Elements, Response,Response Element