Mapping regions of yeast transcription factor IIIA required for DNA binding, interaction with transcription factor IIIC, and transcription activity.
TFIIIA, a site-specific DNA-binding protein containing nine zinc finger motifs, is an RNA polymerase III transcription factor responsible for the assembly of a transcription complex on the 5 S RNA gene. We have analyzed the effect of deleting various regions of yeast TFIIIA on its ability to bind to the internal control region of the yeast 5 S RNA gene, to recruit TFIIIC into the TFIIIA.DNA complex and to promote in vitro transcription of the 5 S RNA gene. A truncated polypeptide containing only the three amino-terminal zinc fingers retained the ability to bind specifically to the internal control region of the 5 S RNA gene. The TFIIIA.DNA complex formed with this three zinc finger module was able to recruit TFIIIC but could not promote transcription. An 81-amino acid domain, which interrupts the repeating zinc finger motifs between fingers 8 and 9, was found to be essential for the transcription activity of the protein. Removal of the carboxyl-terminal region of yeast TFIIIA that extends beyond the ninth zinc finger motif had no effect on the in vitro activities of the protein. Extending the carboxyl-terminal deletion to include the ninth zinc finger led to reduced transcription activity. We speculate that the ninth zinc finger serves to anchor the carboxyl-terminal portion of TFIIIA on the 5 S RNA gene, correctly orienting the 81-amino acid domain to serve its role in promoting transcription. Removal of the region of the protein amino-terminal to the first zinc finger motif was without effect. Extending the amino-terminal deletion to include the first zinc finger appeared to decrease the affinity of TFIIIA for DNA without affecting the specificity of the protein-DNA interaction. The resultant TFIIIA.DNA complex was defective in recruiting TFIIIC and did not support transcription.
Department of Molecular and Medical Genetics, University of Toronto, Ontario, Canada.
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Milne
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Segall
D000595:Amino Acid Sequence; D001483:Base Sequence; D003062:Codon; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D012320:RNA Polymerase III / Q000378:metabolism; D012341:RNA, Ribosomal, 5S / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D019474:Transcription Factors, TFIII; D014158:Transcription, Genetic; D016335:Zinc Fingers / Q000235:genetics / Q000502:physiology; D000098832:Transcription Factor TFIIIC
D003062:Codon; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D012341:RNA, Ribosomal, 5S; D011994:Recombinant Proteins; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors; D019474:Transcription Factors, TFIII; D000098832:Transcription Factor TFIIIC; D012320:RNA Polymerase III