Mechanism of hairpin-duplex conversion for the HIV-1 dimerization initiation site.
We have used the dimerization initiation site of HIV-1 genomic RNA as a model to investigate hairpin-duplex interconversion with a combination of fluorescence, UV melting, gel electrophoresis, and x-ray crystallographic techniques. Fluorescence studies with molecular beacons and crystallization experiments with 23-nucleotide dimerization initiation site fragments showed that the ratio of hairpin to duplex formed after annealing in water essentially depends on RNA concentration and not on cooling kinetics. With natural sequences allowing to form the most stable duplex, and thus also the loop-loop complex (or "kissing complex"), concentrations as low as 3 mum in strands are necessary to obtain a majority of the hairpin form. With a mutated sequence preventing kissing complex formation, a majority of hairpins was even obtained at 80 mum in strands. However, this did not prevent an efficient conversion from hairpin to duplex in the presence of salts. Kinetic considerations are in favor of duplex formation from intermediates involving hairpins engaged in cruciform dimers rather than from free strands. The very first step of formation of such a cruciform intermediate could be trapped in a crystal structure. This mechanism might be significant for the dynamics of small RNAs beyond the strict field of HIV-1.
Bernacchi
Serena
S
Institut de Biologie Moléculaire et Cellulaire, UPR 9002 du CNRS Conventionnée à l'Université Louis Pasteur Strasbourg, F-67084 Strasbourg Cedex, France.
Ennifar
Eric
E
Tóth
Katalin
K
Walter
Philippe
P
Langowski
Jörg
J
Dumas
Philippe
P
D001483:Base Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004586:Electrophoresis; D016679:Genome, Viral; D015497:HIV-1 / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012313:RNA / Q000737:chemistry; D012367:RNA, Viral / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors; D014466:Ultraviolet Rays; D014779:Virus Replication
D012367:RNA, Viral; D012313:RNA