Contribution of individual amino acids to the nucleic acid binding activities of the Xenopus zinc finger proteins TFIIIIA and p43

Abstract

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.