New, extended hairpin form of the TAR-2 RNA domain points to the structural polymorphism at the 5' end of the HIV-2 leader RNA.
The HIV-2 TAR RNA domain (TAR-2) plays a key role in the trans-activation of HIV-2 transcription as it is the target for the Tat-2 protein and several cell factors. Here, we show that the TAR-2 domain exists in vitro in two global, alternative forms: a new, extended hairpin form with two conformers and the already proposed branched hairpins form. This points strongly to the structural polymorphism of the 5' end of the HIV-2 leader RNA. The evidence comes from the non-denaturing PAGE mobility assay, 2D structure prediction, enzymatic and Pb2+- or Mg2+-induced RNA cleavages. Existence of the TAR-2 extended form was further proved by the examination of engineered TAR-2 mutants stabilized either in the branched or extended structure. The TAR-2 extended form predominates with an increasing magnesium concentration. Gel retardation assays reveal that both TAR-2 wt and its mutant, unable to form branched structure, bind Tat-2 protein with comparable, high affinity, while RNA hairpins I and II, derived from TAR-2 branched structure model, show much less protein binding. We propose that an internal loop region of the TAR-2 extended hairpin form is a potential Tat-2 binding site.
Pachulska-Wieczorek
Katarzyna
K
Laboratory of Structural Chemistry of Nucleic Acids, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznań, Poland.
Purzycka
Katarzyna J
KJ
Adamiak
Ryszard W
RW
D020121:5' Untranslated Regions / Q000737:chemistry*; D001120:Arginine / Q000031:analogs & derivatives / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D004047:Diethyl Pyrocarbonate; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D015696:Gene Products, tat / Q000378:metabolism; D016325:HIV Long Terminal Repeat; D015498:HIV-2 / Q000235:genetics*; D007854:Lead / Q000737:chemistry; D008274:Magnesium / Q000737:chemistry; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism; D012260:Ribonucleases; D054322:tat Gene Products, Human Immunodeficiency Virus
D020121:5' Untranslated Regions; D015696:Gene Products, tat; D012367:RNA, Viral; D054322:tat Gene Products, Human Immunodeficiency Virus; C069339:argininamide; D007854:Lead; D001120:Arginine; D012260:Ribonucleases; D008274:Magnesium; D004047:Diethyl Pyrocarbonate