HIV-1 nucleocapsid protein switches the pathway of transactivation response element RNA/DNA annealing from loop-loop "kissing" to "zipper".
The chaperone activity of HIV-1 (human immunodeficiency virus type 1) nucleocapsid protein (NC) facilitates multiple nucleic acid rearrangements that are critical for reverse transcription of the single-stranded RNA genome into double-stranded DNA. Annealing of the transactivation response element (TAR) RNA hairpin to a complementary TAR DNA hairpin is an essential step in the minus-strand transfer step of reverse transcription. Previously, we used truncated 27-nt mini-TAR RNA and DNA constructs to investigate this annealing reaction pathway in the presence and in the absence of HIV-1 NC. In this work, full-length 59-nt TAR RNA and TAR DNA constructs were used to systematically study TAR hairpin annealing kinetics. In the absence of NC, full-length TAR hairpin annealing is approximately 10-fold slower than mini-TAR annealing. Similar to mini-TAR annealing, the reaction pathway for TAR in the absence of NC involves the fast formation of an unstable "kissing" loop intermediate, followed by a slower conversion to an extended duplex. NC facilitates the annealing of TAR by approximately 10(5)-fold by stabilizing the bimolecular intermediate ( approximately 10(4)-fold) and promoting the subsequent exchange reaction ( approximately 10-fold). In contrast to the mini-TAR annealing pathway, wherein NC-mediated annealing can initiate through both loop-loop kissing and a distinct "zipper" pathway involving nucleation at the 3'-/5'-terminal ends, full-length TAR hairpin annealing switches predominantly to the zipper pathway in the presence of saturated NC.
Vo
My-Nuong
MN
Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA.
Barany
George
G
Rouzina
Ioulia
I
Musier-Forsyth
Karin
K
D020029:Base Pairing; D001483:Base Sequence; D004279:DNA, Viral / Q000378:metabolism*; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000502:physiology*; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000378:metabolism*; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*
D004279:DNA, Viral; C071884:NCP7 protein, Human immunodeficiency virus 1; D012367:RNA, Viral; D054301:gag Gene Products, Human Immunodeficiency Virus