The N-terminus of HIV-1 Tat protein is essential for Tat-TAR RNA interaction.
The human HIV transactivator protein Tat is essential for efficient viral transcription that occurs by a complex mechanism involving interaction of Tat with the TAR RNA element. This interaction appears to require the mediation of a cellular protein, cyclin T1. However, the possibility that Tat and TAR associate in a binary Tat-TAR complex has been little investigated. Using a chemically synthesized active Tat protein, the kinetic and equilibrium parameters of its interaction with TAR were determined by surface plasmon resonance technology. Independently of partner and method of immobilization onto the sensor chip, the association (k(a) = 5-9 x 10(5) M(-1) s(-1)) and dissociation rate constants (k(d) = 1.7-4.3 x 10(-3) s(-1)) yielded similar equilibrium dissociation constants (K(d) = 2-8 nM). A truncated peptide encompassing residues 30-86 of Tat did not bind to TAR at all. We conclude that Tat can form a high-affinity complex with TAR in the absence of cyclin T1 and that the N-terminal domain of Tat is essential for this interaction, suggesting a conformational link between this domain and the basic domain of Tat. These results are important in our quest for developing therapeutic compounds that impair viral replication.
Chaloin
O
O
UPR 9021, Institut de Biologie Moléculaire et Cellulaire, CNRS, 15 rue René Descartes, 67084 Strasbourg, France. o.chaloin@ibmc.u-strasbg.fr
Peter
J-C
JC
Briand
J-P
JP
Masquida
B
B
Desgranges
C
C
Muller
S
S
Hoebeke
J
J
D015696:Gene Products, tat / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007103:Immobilization; D007700:Kinetics; D040081:Protein Array Analysis; D011485:Protein Binding / Q000502:physiology; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance; D013997:Time Factors
D015696:Gene Products, tat; D012367:RNA, Viral; D011994:Recombinant Proteins; D019809:Streptavidin
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