Figure 6.

Schematic representation of P-TEFb–Tat-TAR ternary complex. P-TEFb is a two-subunit kinase complex containing a CDK9 and a CycT1. CDK9 subunit of P-TEFb binds to the cyclin box sequence (amino acids 1–250) of CycT1. A Tat:TAR recognition motif (TRM) in the CycT1 sequence spans amino acid residues 251–272, which is necessary to form a complex with Tat and TAR RNA. Amino acid sequence 252–260 from TRM was cross-linked to the U31 side of the TAR loop, suggesting that the residues 261–272 are involved in interaction with Tat core domain (amino acids 1–48), shown as a round ball indicating a compact structure. RNA is recognized by the Arg-rich RNA recognition motif (ARM) of Tat, and Lys-50 interacts with the G34 side of the TAR loop. CycT1 and Tat binding to TAR RNA is highly cooperative and a capacity of 85%, Hill coefficient of 2.7, and dissociation constant (K_D) of 2.45 nM were observed (18), indicating that there are three binding sites on TAR RNA. It is conceivable that the CycT1–Tat heterodimer directly binds to TAR RNA in the U-rich RNA bulge region, and this binding facilitates the interactions of CycT1–Tat at the other two sites in the RNA loop: CycT1 interacts with the U31 side, and Tat binds to the G34 side. Amino acids cross-linked with TAR RNA are shown by solid arrows and the suggested Tat-interacting region of TRM is shown by a broken arrow. CycT1 is shown in cyan, CDK9 in blue, and Tat in pink. N and C termini of proteins are labeled as NH₂ and COOH, respectively.