Recognition of the high affinity binding site in rev-response element RNA by the human immunodeficiency virus type-1 rev protein.
The Human Immunodeficiency Virus type-1 rev protein binds with high affinity to a bubble structure located within the rev-response element (RRE) RNA in stemloop II. After this initial interaction, additional rev molecules bind to the RRE RNA in an ordered assembly process which requires a functional bubble structure, since mutations in the bubble sequence that reduce rev affinity block multiple complex formation. We have used synthetic chemistry to characterize the interaction between rev protein and its high affinity binding site. A minimal synthetic duplex RNA (RBC6) carrying the bubble and 12 flanking base pairs is able to bind rev with 1 to 1 stoichiometry and with high affinity. When the bubble structure is inserted into synthetic RNA molecules carrying longer stretches of flanking double-stranded RNA, rev forms additional complexes resembling the multimers observed with the RRE RNA. The ability of rev to bind to RBC6 analogues containing functional group modifications on base and sugar moieties of nucleoside residues was also examined. The results provide strong evidence that the bubble structure contains specific configurations of non-Watson--Crick G:G and G:A base pairs and suggest that high affinity recognition of RRE RNA by rev requires hydrogen bonding to functional groups in the major groove of a distorted RNA structure.
D017123:Bacteriophage T7 / Q000201:enzymology; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D015694:Gene Products, rev / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006860:Hydrogen Bonding; D007202:Indicators and Reagents; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D012313:RNA / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D012260:Ribonucleases; D014158:Transcription, Genetic; D054321:rev Gene Products, Human Immunodeficiency Virus
D015694:Gene Products, rev; D007202:Indicators and Reagents; D046911:Macromolecular Substances; D009843:Oligoribonucleotides; D054321:rev Gene Products, Human Immunodeficiency Virus; D012313:RNA; D012321:DNA-Directed RNA Polymerases; D012260:Ribonucleases