Transcriptional activity of transcription factor IIE is dependent on zinc binding.
The functions of individual basal transcription factors during the formation of an initiation complex by RNA polymerase II remain largely unknown. Transcription factor IIE (TFIIE) has recently been shown to bind to multiple targets in the initiation complex. To assess the role of zinc binding in basal transcription, we have mutated the predicted zinc-finger domain of human TFIIE. Atomic absorption spectroscopy using purified recombinant proteins revealed that the large subunit, TFIIE-56, is indeed a zinc-binding protein. Mutation of a cysteine residue in the putative zinc-finger domain abolished zinc binding. Moreover, mutant TFIIE-56 failed to support reconstituted basal transcription in vitro, suggesting that zinc binding is required for TFIIE function. However, gel-filtration experiments and protein affinity experiments suggest that mutant TFIIE-56 forms a stable heterotetramer with the small subunit, TFIIE-34, that is similar to wild type. Interestingly, gel mobility shift experiments reveal that loss of transcriptional activity by mutant TFIIE is correlated with its inability to stably assemble into the transcription complex. These findings establish that zinc binding by TFIIE may help form a specific structure that is required for stable entry into the transcription complex.
Maxon
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Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Tjian
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D000409:Alanine; D000595:Amino Acid Sequence; D001483:Base Sequence; D003545:Cysteine; D004268:DNA-Binding Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D016385:TATA Box; D035181:TATA-Box Binding Protein; D035581:Transcription Factor TFIIB; D014157:Transcription Factors / Q000378:metabolism*; D019473:Transcription Factors, TFII; D014158:Transcription, Genetic; D015032:Zinc / Q000378:metabolism*; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D035181:TATA-Box Binding Protein; D035581:Transcription Factor TFIIB; D014157:Transcription Factors; D019473:Transcription Factors, TFII; C066644:transcription factor TFIIE; D015032:Zinc; D003545:Cysteine; D000409:Alanine