A structural model for the HIV-1 Rev-RRE complex deduced from altered-specificity rev variants isolated by a rapid genetic strategy.
A broadly applicable genetic strategy was developed for investigating RNA-protein interactions and applied to the HIV-1 Rev protein. By rapidly screening thousands of Rev-RNA interactions in Escherichia coli, we isolated Rev suppressor mutations that alleviated the deleterious effect of mutations in RRE stem-loop IIB, the high affinity RNA-binding site for Rev. All of these suppressor mutations map to a single arginine-deficient face of a Rev alpha-helix, and some alter the binding specificity of the protein, providing genetic evidence for direct contacts between specific Rev amino acids and RNA nucleotides in the RNA complex of Rev. The spatial constraints suggested by these data have enabled us to model the structure of this complex.
Jain
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Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Belasco
J G
JG
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000502:physiology; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015694:Gene Products, rev / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005821:Genetic Techniques; D015497:HIV-1; D006801:Humans; D007763:Lac Operon; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D014176:Protein Biosynthesis; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D013489:Suppression, Genetic; D054321:rev Gene Products, Human Immunodeficiency Virus
D000596:Amino Acids; D015694:Gene Products, rev; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus