RNA binding assays for Tat-derived peptides: implications for specificity.
RNA recognition by the HIV Tat protein is mediated in part by an arginine- and lysine-rich basic subdomain implicated as a signature element in proteins that bind RNA. Relative RNA binding affinities for a 14-residue peptide derived from Tat that spans the basic region are determined using a competition protocol. Binding specificity is compared with complexation by a 38-residue model for the RNA binding domain of Tat using the same approach. Binding strength for the minimal (14 residue) peptide is correlated with that for the longer peptide: both peptides recognize a short, bulged duplex. However, the shorter peptide dissociates more rapidly from the wild-type site and discriminates less well between nonspecific (double-stranded RNA) and specific sites. Relative dissociation constants for 38-residue peptide determined from direct partition and competition assays differ; the former assay consistently predicts stronger discrimination against RNAs with mutations in the stems flanking the bulge. Differences between the two assays are reconciled in terms of contributions from labile binding which is unstable to native gel electrophoresis. Kinetic stability may constitute a major specificity determinant for basic subdomain-mediated recognition of RNA.
Weeks
K M
KM
Department of Chemistry, Yale University, New Haven, Connecticut 06511.
Crothers
D M
DM
D001483:Base Sequence; D001667:Binding, Competitive; D015696:Gene Products, tat / Q000378:metabolism*; D006678:HIV / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000378:metabolism*; D009994:Osmolar Concentration; D012313:RNA / Q000138:chemical synthesis / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D054322:tat Gene Products, Human Immunodeficiency Virus; D000099253:Trans-Activation Responsive RNA-Binding Protein
D015696:Gene Products, tat; D009843:Oligoribonucleotides; D012313:RNA; D016601:RNA-Binding Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D000099253:Trans-Activation Responsive RNA-Binding Protein