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. 1985 Jul 25;13(14):5369–5387. doi: 10.1093/nar/13.14.5369

Characterization of the RNA binding properties of transcription factor IIIA of Xenopus laevis oocytes.

P J Romaniuk
PMCID: PMC321871  PMID: 2410862

Abstract

A nitrocellulose filter binding assay has been developed to study the interaction of Xenopus transcription factor IIIA with 5S RNA. The protein binds Xenopus oocyte 5S RNA with an association constant of 1.4 X 10(9) M-1 at 0.1 M salt, pH 7.5 at 20 degrees C. TF IIIA binds wheat germ 5S RNA with a two-fold higher affinity, E. coli 5S RNA with a four-fold weaker affinity, and has a barely detectable interaction with yeast tRNAphe. The preference for binding eukaryotic 5S RNA is enhanced in competition assays. The homologous reconstituted complex contains one molecule each of protein and 5S RNA and is indistinguishable from native 7S RNP in mobility on non-denaturing polyacrylamide gels. The conformation of the RNA in reconstituted particles is identical to the conformation of RNA in native 7S RNP. Further analysis of the homologous interaction reveals that complex formation is a favoured both by enthalpy and entropy. The 5S RNA binding activity has a broad pH optimum spanning pH 6.0 to pH 8.0. Determination of the salt dependence of Ka reveals that as many as 5 lysine-phosphate type ionic bonds may be formed in the homologous complex. Approximately 68% of the free energy of complex formation is contributed by non-electrostatic interactions between TF IIIA and Xenopus 5S RNA.

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Selected References

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