title: Analysis of arginine-rich peptides from the HIV Tat protein reveals unusual features of RNA-protein recognition.
abstract: Arginine-rich sequences are found in many RNA-binding proteins and have been proposed to mediate specific RNA recognition. Fragments of the HIV-1 Tat protein that contain the arginine-rich region of Tat bind specifically to a 3-nucleotide bulge in TAR RNA. To determine the amino acid requirements for specific RNA recognition, we synthesized a series of mutant Tat peptides spanning this domain (YGRKKRRQRRRP) and measured their affinity and specificity for TAR RNA. Several corresponding mutations were introduced into the full-length Tat protein, and trans-activation activity was measured. Systematic substitution of arginine residues with alanines or lysines suggested that overall charge density is important but did not point to any specific residues as being essential for binding. A glutamine-to-alanine substitution had no effect on binding. Remarkably, peptides with scrambled or reversed sequences showed the same affinity and specificity for TAR RNA as the wild-type peptide. Trans-activation activity of the mutant Tat proteins correlated with RNA binding. Arginine-rich peptides from SIV Tat and from HIV-1 Rev, which can functionally substitute for the basic region of HIV-1 Tat, also bound specifically to TAR. Circular dichroism spectra suggest that the arginine-rich region of Tat is unstructured in the absence of RNA, becomes partially or fully structured upon binding, and induces a conformational change in the RNA. These results suggest that arginine-rich RNA-binding domains have considerable sequence flexibility, reminiscent of acidic domains found in transcriptional activators, and that RNA structure may provide much of the specificity for the interaction.
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142.
authors.forename: B J
authors.initials: BJ
authors.lastname: Calnan
authors.forename: S
authors.initials: S
authors.lastname: Biancalana
authors.forename: D
authors.initials: D
authors.lastname: Hudson
authors.forename: A D
authors.initials: AD
authors.lastname: Frankel
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism*; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D002942:Circular Dichroism; D015696:Gene Products, tat / Q000032:analysis / Q000378:metabolism*; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000378:metabolism*; D016601:RNA-Binding Proteins; D015533:Transcriptional Activation; D054322:tat Gene Products, Human Immunodeficiency Virus
chemical_list: D002352:Carrier Proteins; D015696:Gene Products, tat; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D001120:Arginine