Characterization of the 5 S RNA binding activity of Xenopus zinc finger protein p43.
One major component of the Xenopus 42 S ribonucleoprotein (RNP) storage particle is the p43 protein. The 5 S RNA binding protein is structurally similar to TFIIIA, containing nine zinc finger domains. The RNA binding properties of recombinant p43 were characterized using a nitrocellulose filter binding assay. The experimental conditions necessary for in vitro p43-5 S RNA complex formation include: pH 7.5, 0.1 M KCl and incubation at 22 degrees C. Under these conditions, the protein binds to Xenopus oocyte 5 S RNA with an apparent association constant of 1.61(+/- 0.12) x 10(9) M-1. A series of mutations in 5 S RNA were used to determine which sequence and structural features of the 5 S RNA are required for high affinity binding of p43. The primary contact points for p43 include the sequences and structures of stems II, V and loop D of the 5 S RNA. Although p43 and TFIIIA are structurally similar and are both relatively insensitive to mutations in the 5 S RNA, they do require different features of the 5 S RNA molecule for high affinity binding.
Department of Biochemistry and Microbiology, University of Victoria, B.C., Canada.
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Romaniuk
D000818:Animals; D001483:Base Sequence; D002413:Cations, Divalent; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011485:Protein Binding; D012341:RNA, Ribosomal, 5S / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012269:Ribosomal Proteins; D012492:Salts; D013696:Temperature; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*; D014981:Xenopus; D029867:Xenopus Proteins; D016335:Zinc Fingers
D002413:Cations, Divalent; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C489267:LOC397745 protein, Xenopus; D012341:RNA, Ribosomal, 5S; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; D012492:Salts; D014157:Transcription Factors; D029867:Xenopus Proteins