The number of positively charged amino acids in the basic domain of Tat is critical for trans-activation and complex formation with TAR RNA.
The basic domain of Tat is required for trans-activation of viral gene expression. We have performed scanning peptide studies to demonstrate that only this domain is capable of binding to the TAR RNA stem-loop. Strikingly, the basic domain of the other human immunodeficiency virus trans-acting factor, Rev, but no other region, is also capable of binding to TAR. Peptide derivatives of Tat do not require the highly conserved glutamine residue at position 54 for TAR binding, since it may be substituted or deleted. In addition, the two lysine residues may be replaced by arginines. Analysis of binding and trans-activation demonstrated that homopolymers of arginine can completely substitute for the basic domain. Such homopolymers have high affinity for wild-type TAR RNA and lower affinity for mutant TAR. Homopolymers of six to nine arginines substituting for the basic domain of Tat enable full trans-activation in vivo. Homopolymers of at least seven arginines are required for detectable in vitro complex formation, although approximately 30% trans-activation is achieved with a mutant Tat containing only five arginines.
Delling
U
U
Department of Biochemistry, McGill University, Canada.
Roy
S
S
Sumner-Smith
M
M
Barnett
R
R
Reid
L
L
Rosen
C A
CA
Sonenberg
N
N
D000595:Amino Acid Sequence; D001120:Arginine; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D015694:Gene Products, rev / Q000235:genetics / Q000378:metabolism; D015696:Gene Products, tat / Q000235:genetics* / Q000378:metabolism; D015756:Genes, tat; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012367:RNA, Viral / Q000235:genetics*; D015533:Transcriptional Activation; D014162:Transfection; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus
D015694:Gene Products, rev; D015696:Gene Products, tat; D012367:RNA, Viral; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus; D001120:Arginine; D015500:Chloramphenicol O-Acetyltransferase