Structural analysis of wild-type and mutant human immunodeficiency virus type 1 Tat proteins.
We expressed the human immunodeficiency virus type 1 transactivator protein, Tat, in the wheat germ cell-free translation system and found it to exist as a monomer. The first coding exon (residues 1 to 72) of wheat germ-expressed Tat was resistant to trypsin digestion, indicating that it is a highly folded, independently structured protein domain. Several mutant Tat proteins were dramatically more sensitive to trypsin than the wild type was, suggesting that their reduced transactivation activities are the result of destabilized structures. Mutant proteins with single-amino-acid substitutions were also identified that had reduced transactivation activities but wild-type structures in the trypsin assay. These mutants clustered in two regions of Tat, at acidic residues 2 and 5 in the amino terminus and between residues 18 and 32. These mutants, wild type in structure but reduced in activity, identify residues in the wild-type protein that may directly contact other molecules during Tat function.
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Cold Spring Harbor Laboratory, New York 11724.
Carlotti
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D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D005091:Exons; D015696:Gene Products, tat / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015756:Genes, tat; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D010957:Plasmids; D014176:Protein Biosynthesis; D015533:Transcriptional Activation; D014357:Trypsin / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D054322:tat Gene Products, Human Immunodeficiency Virus; D014357:Trypsin; D000409:Alanine