Circular dichroism studies suggest that TAR RNA changes conformation upon specific binding of arginine or guanidine.
Short basic peptides from the HIV Tat protein bind specifically to a bulge region in TAR RNA, with a single arginine residue providing the only sequence-specific contact. The free amino acid arginine also binds specifically to TAR. Previous circular dichroism (CD) experiments suggested that peptide binding induces a conformational change in TAR. Here we confirm this observation using single arginine-containing peptides and show that arginine or guanidine binding also induces a conformational change in TAR. A peptide containing a single arginine within a stretch of histidines (CYHHHRHHHHHA) shows pH-dependent binding and a corresponding change in TAR conformation, as detected by a decrease in the CD signal at 265 nm. Arginine and guanidine, which bind to TAR with apparent Kd's of approximately 1.5 mM, induce similar CD changes. In contrast, lysine, which does not bind specifically to TAR, has no effect. Mutants of TAR that abolish specific binding (a U-->C substitution in the three-nucleotide bulge, a deletion of the bulge, or an A-U to U-A base pair change above the bulge) show no change in the CD signal upon binding of peptides, arginine, or guanidine. The results suggest that binding of a single guanidinium group to a specific site in TAR induces a change in RNA conformation.
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Whitehead Institute for Biomedical Research, Nine Cambridge Center, Massachusetts 02142.
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D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism*; D017123:Bacteriophage T7 / Q000201:enzymology; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D015696:Gene Products, tat / Q000378:metabolism*; D019791:Guanidine; D006146:Guanidines / Q000378:metabolism*; D006678:HIV / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D012313:RNA / Q000737:chemistry* / Q000378:metabolism*; D014158:Transcription, Genetic; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D006146:Guanidines; D010455:Peptides; D054322:tat Gene Products, Human Immunodeficiency Virus; D012313:RNA; D001120:Arginine; D012321:DNA-Directed RNA Polymerases; D019791:Guanidine