CLONING AND CHARACTERIZATION OF HTAF(II)18, HTAF(II)20 AND HTAF(II)28 - 3 SUBUNITS OF THE HUMAN TRANSCRIPTION FACTOR TFIID

We have cloned cDNAs encoding three novel TAF(II)s [TATA-binding protein (TBP)-associated factors] from the human (h) HeLa cell TFIID complexes hTAF(II)28, hTAF(II)20 and hTAF(II)18. hTAF(II)28 is a core hTAF(II) present in both of the previously described hTFIID species which either lack or contain hTAF(II)30 (hTFIID alpha and hTFIID beta-respectively), and is the homelogue of Drosophila (d)TAF(II)30 beta hTAF(II)18 is a novel hTAF(II) which shows homology to the N-terminal region of the yeast TAF(II) SPT3, but has no known Drosophila counterpart. In contrast to hTAF(II)28, hTAF(II)18 is a TFIID beta-specific hTAF(II). hTAF(II)20 is the homologue of p22, an alternatively spliced form of dTAF(II)30 alpha (p32). Using a combination of protein affinity chromategraphy and cotransfection and immunoprecipitation assays, we have identified a series of in vitro and intracellular interactions among the novel hTAF(II)s and between the novel hTA(II)ns and hTAF(II)30 or TBP. We show that hTAF(II)28 interacts with hTAF(II)18 both in vitro and intracellularly; in contrast to its Drosophila homologue, hTAF(II)28 also interacts directly with TBP. Deletion analysis indicates that TBP and hTAF(II)18 bind to distinct domains of hTAF(II)28. hTAF(II)18 also interacts with TBP, but it interacts more strongly with hTAF(II)28 and hTAF(II)30. The binding of hTAF(II)28 and hTAF(II)30 requires distinct domains of hTAF(II)28. As observed with the homologous Drosophila proteins, hTAF(II)20 interacts directly with TBP; however, additional interactions between hTAF(II)20 and hTAF(II)28 or hTAF(II)30 were detected. These results reveal differences not only in subunit composition, but also in the organization of dTFIID and hTFIID complexes.