Orientation-specific cis complementation by bulge- and loop-mutated human immunodeficiency virus type 1 TAR RNAs.
Tat activates human immunodeficiency type 1 gene expression by binding to TAR RNA. TAR comprises a partially base paired stem and hexanucleotide loop with a tripyrimidine bulge in the upper stem. In vitro, Tat binds to the bulge and upper stem, with no requirement for the loop. However, in vivo, loop sequences are critical for activation, implying that a loop binding cellular factor may be involved in the activation pathway. Given that activation appears to be a two-component system comprising a Tat-bulge interaction and a cellular factor-loop interaction, we considered that it might be possible to spatially separate the two components and retain activation. We have constructed a series of double TAR elements comprising various combinations of mutated TAR structures. Defective TARs with nucleotide substitutions in either the bulge or the loop complemented each other to give wild-type activation. However, the complementation was orientation specific, requiring the intact Tat binding site to reside on the 5'-proximal TAR. These data suggest that provided the wild-type orientation of the bulge and loop elements is retained, there is no requirement for them to coexist on the same TAR structure.
Department of Biochemistry, University of Oxford, United Kingdom.
M
M
Braddock
R
R
Powell
J
J
Sutton
A J
AJ
Kingsman
S M
SM
Kingsman
D001426:Bacterial Proteins / Q000096:biosynthesis*; D001483:Base Sequence; D001665:Binding Sites; D002628:Chemoreceptor Cells; D029968:Escherichia coli Proteins; D015696:Gene Products, tat / Q000378:metabolism; D005816:Genetic Complementation Test; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis*; D008961:Models, Structural; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D011956:Receptors, Cell Surface; D054322:tat Gene Products, Human Immunodeficiency Virus
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D015696:Gene Products, tat; D008565:Membrane Proteins; D012367:RNA, Viral; D011956:Receptors, Cell Surface; C057331:Tar protein, E coli; D054322:tat Gene Products, Human Immunodeficiency Virus