This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Transcription activation by binding of the Human Immunodeficiency Virus type-1 (HIV-1) transactivator (Tat) protein to the Tat-responsive RNA (TAR) element is essential for viral replication and is considered a potential target for antiviral drugs. An arginine rich motif (RKKRRQRRR) mediates TAR RNA recognition by Tat. However, previous studies suggest that this short basic stretch is insufficient to determine the entire binding activity of Tat, since amino acids outside of this basic region also contribute to this interaction. To gain a better understanding of the functional and structural determinants that mediate Tat-TAR interaction, we used deep mutational scanning with a yeast three-hybrid system. Deep mutational scanning is an approach that queries the fitness effects of mutations in every position in a protein domain of interest. In this assay, Tat-TAR interaction creates a functional transcription factor that activates the expression of the HIS3 reporter gene, allowing yeast cells to grow under histidine starvation conditions. We determined that a segment of 40 amino acids within the Tat protein is necessary for TAR binding and created a library of Tat genes, encompassing ~106 different mutants, engineered to have an average of 2.5 random mutations in this region. The effect of each Tat mutation on TAR binding determines the induction level of the HIS3 reporter gene in each tranformant and hence, its growth rate under histidine deficient conditions.
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