Binding of phage-displayed HIV-1 Tat to TAR RNA in the presence of cyclin T1.
The transactivator protein (Tat) of the human immunodeficiency virus (HIV) is a key regulatory protein in the viral replication cycle. Together with cellular cyclin T1 and an RNA element (transactivation response; TAR) located at the 5' end of all viral transcripts, it forms a ternary complex that ultimately enhances the expression of all viral genes. In this ternary complex, cyclin T1 interacts directly with Tat and TAR. The presence of cyclin T1 is essential for high TAR RNA affinity and specificity of Tat. To study protein-protein and protein-RNA interaction, we developed a phage display system that displays functional Tat on the surface of bacteriophage M13. The addition of recombinant cyclin T1 to the selections yielded a phage display system that mirrors all binding properties of the cyclin T1-Tat-TAR complex known from cell assays and biochemical studies. Phage-displayed Tat protein as well as the cyclin T1 are fully functional. The relative binding capabilities of wild-type- and mutant Tat-displaying phages show that the presence of cyclin T1 significantly reduces the importance of basic residues in the basic sequence region of Tat for its binding to TAR.
Jonas
G
G
Institut für Molekulare Biotechnologie, Jena, Deutschland.
Hoffmann
S
S
Willbold
D
D
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002213:Capsid / Q000235:genetics / Q000378:metabolism; D036022:Capsid Proteins; D056906:Cyclin T; D016213:Cyclins / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D015696:Gene Products, tat / Q000378:metabolism*; D016325:HIV Long Terminal Repeat; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
C503684:CCNT1 protein, human; D036022:Capsid Proteins; D056906:Cyclin T; D016213:Cyclins; D015696:Gene Products, tat; D008565:Membrane Proteins; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; C025668:coat protein, Bacteriophage M13