Interaction of human immunodeficiency virus type 1 Tat-derived peptides with TAR RNA.
Basic peptides from the carboxy terminus of the human immunodeficiency virus type 1 (HIV-1) Tat protein bind to the stem-loop region of TAR RNA, spanning a trinucleotide bulge, with high affinity and moderate specificity. Previous studies have demonstrated that TAR RNA contains a specific arginine binding pocket. A series of 24 amino acid Tat-derived peptides with one or two arginines has been evaluated as possible structural models of the wild-type peptide in its interaction with TAR RNA, using gel electrophoretic methods and circular dichroism (CD) spectroscopy. Dissociation rate measurements indicate that these peptides form complexes with TAR RNA that are significantly less stable kinetically than the wild-type complex. Through a combination of dissociation and association rate measurements, we estimate that wild-type Tat peptide and TAR RNA interact with a Kd of about 16 pM. Together with competition experiments, these results confirm that band shift gel titration methods significantly underestimate absolute peptide-RNA binding affinities in the subnanomolar range. Through competition experiments with bulge mutants of TAR RNA, we demonstrate that peptides that form longer lived complexes with wild-type TAR RNA also show greater discrimination over TAR RNA bulge mutants. Difference CD spectra show that the Tat-derived peptides do not induce the same changes in TAR RNA as the wild-type peptide. The difference CD spectrum of argininamide bound to TAR RNA is most similar to that of the wild-type peptide-TAR RNA complex, implying that the differences in CD spectra upon complex formation are mostly due to changes in TAR RNA conformation.
Long
K S
KS
Department of Chemistry, Yale University, New Haven, Connecticut 06511, USA.
Crothers
D M
DM
D000595:Amino Acid Sequence; D001483:Base Sequence; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism*; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D010446:Peptide Fragments; D012367:RNA, Viral; D054322:tat Gene Products, Human Immunodeficiency Virus