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 prote in (TBP)-associated factors] from the human (h) HeLa cell TFIID comple xes hTAF(II)28, hTAF(II)20 and hTAF(II)18. hTAF(II)28 is a core hTAF(I I) present in both of the previously described hTFIID species which ei ther lack or contain hTAF(II)30 (hTFIID alpha and hTFIID beta-respecti vely), 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 co ntrast to hTAF(II)28, hTAF(II)18 is a TFIID beta-specific hTAF(II). hT AF(II)20 is the homologue of p22, an alternatively spliced form of dTA F(II)30 alpha (p32). Using a combination of protein affinity chromateg raphy and cotransfection and immunoprecipitation assays, we have ident ified a series of ill vitro and intracellular interactions among the n ovel 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 in tracellularly; in contrast to its Drosophila homologue, hTAF(II)28 als o interacts directly with TBP. Deletion analysis indicates that TBP an d hTAF(II)18 bind to distinct domains of hTAF(II)28. hTAF(II)18 also i nteracts 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 pro teins, hTAF(II)20 interacts directly with TBP; however, additional int eractions between hTAF(II)20 and hTAF(II)28 or hTAF(II)30 were detecte d. These results reveal differences not only in subunit composition, b ut also in the organization of dTFIID and hTFIID complexes.