Some TAF subunits of transcription factor TFIID play a pivotal role in transcriptional activation by mediating protein-protein interactions, whereas other TAFs direct promoter selectivity via protein-DNA recognition. Here, we report that purified recombinant TAFII250 is a protein serine kinase that selectively phosphotylates RAP74 but not other basal transcription factors or common phosphoacceptor proteins. The phosphorylation of RAP74 also occurs in the context of the complete TFIID complex. Deletion analysis revealed that TAFII250 contains two distinct kinase domains each capable of autophosphorylation. However, both the N- and C-terminal kinase domains of TAFII250 are required for efficient transphosphorylation of RAP74 on serine residues. These findings suggest that the targeted phosphorylation of RAP74 by TAFII250 may provide a mechanism for signaling between components within the initiation complex to regulate transcription.

We previously characterized Drosophila and human TAF subunits that make up the core TFIID complex found in all cells. Here, we report that differentiated B cells contain a novel substoichiometric TAF of 105 kDa not found associated with TFIID isolated from other cell types. The cDNA encoding hTAFII105 reveals a highly conserved C-terminal domain shared by hTAFII130 and oTAFII110, while the N-terminal coactivator domain has diverged significantly. All cells tested express TAFII105 mRNA, but only B cells contain significant levels of protein associated with TFIID. Transient overexpression of hTAFII105 selectively squelches the transcription of some genes in B cells. These properties suggest that TAFII105 is a cell type-specific subunit of TFIID that may be responsible for mediating transcription by a subset of activators in B cells.

Translation Initiator of Short 5′ UTR (TISU) is a unique regulatory element of both transcription and translation initiation. It is present in a sizable number of genes with basic cellular functions and a very short untranslated region (5′ UTR). Here, we investigated translation initiation from short 5′ UTR mRNAs with AUG in various contexts. Reducing 5′ UTR length to the minimal functional size increases leaky scanning from weak and strong initiators but hardly affects translation initiation and ribosomal binding directed by TISU. Ribosome interaction with TISU mRNA is cap dependent and involves AUG downstream nucleotides that compensate for the absent 5′ UTR contacts. Interestingly, eIF1 inhibits cap-proximal AUG selection within weak or strong contexts but not within TISU. Furthermore, TISU-directed translation is unaffected by inhibition of the RNA helicase eIF4A. Thus, TISU directs efficient cap-dependent translation initiation without scanning, a mechanism that would be advantageous when intracellular levels of eIF1 and eIF4A fluctuate.