Contribution of individual amino acids to the nucleic acid binding activities of the Xenopus zinc finger proteins TFIIIIA and p43.
Xenopus transcription factor IIIIA (TFIIIA) binds to both 5S RNA and the 5S RNA gene in immature oocytes, an interaction mediated by nine zinc fingers. To determine the role of the central zinc fingers of the protein in these nucleic acid binding activities, a series of substitution mutants of TFIIIA were constructed and expressed as recombinant proteins in Escherichia coli. The mutant proteins were purified to homogeneity and analyzed for DNA and RNA binding activities using a nitrocellulose filter binding assay. Finger 5, but not finger 4, 6, or 7, is involved in sequence-specific binding to the 5S RNA gene. A TWT amino acid motif in finger 6 makes a significant contribution to the binding of TFIIIA to 5S RNA, while mutations in fingers 4, 5, and 7 have little or no effect on RNA binding by TFIIIA. In striking contrast, a TWT motif in finger 6 of p43, another Xenopus zinc finger protein that binds to 5S RNA, is not necessary for 5S RNA binding by this protein. Evidence for the presence of inhibitory finger-finger interactions that limit the nucleic acid binding properties of individual zinc fingers within TFIIIA and p43 is discussed.
Hamilton
T B
TB
Department of Biochemistry and Microbiology, University of Victoria, P.O. Box 3055, Victoria, BC, Canada V8W 3P6.
Turner
J
J
Barilla
K
K
Romaniuk
P J
PJ
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D016162:Genes, Wilms Tumor / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D012341:RNA, Ribosomal, 5S / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012269:Ribosomal Proteins; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D025721:WT1 Proteins; D014981:Xenopus / Q000235:genetics / Q000378:metabolism*; D029867:Xenopus Proteins; D016335:Zinc Fingers / Q000235:genetics
D000596:Amino Acids; D004268:DNA-Binding Proteins; C489267:LOC397745 protein, Xenopus; D010446:Peptide Fragments; D012341:RNA, Ribosomal, 5S; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors; D025721:WT1 Proteins; D029867:Xenopus Proteins