A selection system to study protein-RNA interactions: functional display of HIV-1 Tat protein on filamentous bacteriophage M13.
The transactivator protein (Tat) of the human immunodeficiency virus (HIV) is a key regulatory protein in the viral replication cycle and belongs to the RNA binding proteins of the arginine-rich motif (ARM) family. Very little is known about their mechanism of RNA recognition. To study the principles of RNA-protein recognition we constructed a system to display HIV-1 Tat on the surface of the filamentous bacteriophage M13. HIV-1 Tat (1-72) and a mutant Tat lacking five cysteine residues were cloned into the pAK phagemid system, which allows fusion of the tat gene to a supershort version of the gene for minor M13 coat protein. Expression of the resulting fusion proteins was shown via western blot analysis. Phages displaying functional Tat could be selected from phages without Tat or with a non-functional Tat variant via binding to biotinylated TAR using streptavidin coated paramagnetic beads. By randomizing certain amino acid positions of Tat and screening of the resulting phage libraries for affinity and specificity, we are now able to study the role and importance of amino acids of HIV-1 Tat for affinity and specificity to TAR RNA.
Hoffmann
S
S
Universität Bayreuth, Lehrstuhl für Biopolymere, Germany.
Willbold
D
D
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis; D017104:Bacteriophage M13 / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002213:Capsid / Q000096:biosynthesis / Q000235:genetics; D002628:Chemoreceptor Cells; D003001:Cloning, Molecular; D029968:Escherichia coli Proteins; D015696:Gene Products, tat / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D015756:Genes, tat; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D015696:Gene Products, tat; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D012329:RNA, Bacterial; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C057331:Tar protein, E coli; D054322:tat Gene Products, Human Immunodeficiency Virus