Functional analysis of the human TAFII250 N-terminal kinase domain.
The largest subunit of the human transcription factor TFIID, TAFII250, was previously reported to contain serine/threonine kinase domains that can autophosphorylate and transphosphorylate the large subunit of the basal factor TFIIF. Here, we identify the regions of the N-terminal kinase domain (amino acids 1-414) necessary for kinase activity and examine its function in vivo. Point mutations within two patches of amino acids in the kinase domain decrease both autophosphorylation and transphosphorylation activities. Importantly, we find that TAFII250-bearing mutations within the N-terminal kinase domain exhibit a significantly reduced ability to rescue ts13 cells that express a temperature-sensitive TAFII250. Moreover, transcription from the cyclin A and cdc2 promoters becomes impaired when cotransfected with hTAFII250 containing inactive forms of the N-terminal kinase domain. Our results suggest that the TAFII250 kinase activity is required to direct transcription of at least some genes in vivo.
O'Brien
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T
Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley 94720, USA.
Tjian
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R
D002460:Cell Line; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic; D051548:Histone Acetyltransferases; D006801:Humans; D016296:Mutagenesis / Q000502:physiology; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017354:Point Mutation / Q000502:physiology; D017434:Protein Structure, Tertiary; D035381:TATA-Binding Protein Associated Factors; D013696:Temperature; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D035362:Transcription Factor TFIID; D014158:Transcription, Genetic / Q000502:physiology*; D014162:Transfection
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D035381:TATA-Binding Protein Associated Factors; D015534:Trans-Activators; D035362:Transcription Factor TFIID; D051548:Histone Acetyltransferases; C069001:TATA-binding protein associated factor 250 kDa