Contribution of Individual Amino Acids to the 5S RNA Binding Activity of the Xenopus Zinc Finger Protein p43

Abstract

Xenopus zinc finger protein p43 binds to 5S RNA in immature oocytes to form a 42S ribonucleoprotein storage particle. To determine the role of individual zinc fingers of the protein in this RNA binding activity, a series of deletion and substitution mutants of p43 were constructed. The effects of the various mutations on the RNA binding activity of p43 were determined using a quantitative equilibrium binding assay. The results indicate that zinc fingers 1 and 4 of p43 are essential for the binding of the protein to 5S RNA. In the case of finger 1, four amino acids key to RNA binding are found on the same face of the alpha-helix, while in the case of finger 4, two key residues are clustered at the start of the alpha-helix. The similarities and differences in the mechanisms by which fingers 1 and 4 of p43 interact with 5S RNA are compared to the interaction of the zinc fingers of Xenopus transcription factor IIIA with 5S RNA.

An RNA Aptamer with High Affinity and High Specificity for the 5S RNA Binding Zinc Finger Proteins TFIIIA and p43

“…Comparison of the Effects of Mutations in TFIIIA and p1-4 on Binding to RNA22 and 5S RNA. Previous studies of the interactions of TFIIIA and p43 with 5S RNA identified key amino acids through the analysis of the effects of site-directed mutations in the proteins on RNA binding affinities (26,31,33). The affinities of these same mutant TFIIIA and p43 proteins for RNA22 were measured using the equilibrium binding assay so that the effects of the mutations on RNA22 binding could be compared with the previously obtained data for 5S RNA binding.…”

“…A similar analysis was previously undertaken to identify key residues in the first four fingers of p43 involved in binding to 5S RNA (33), and those mutant proteins were assayed for effects on affinity for RNA22 (Figure 7). Individual finger swap mutations pW1, pW2, pW3, and pW4 previously identified zinc fingers 1 and 4 as being particularly critical for high-affinity binding to 5S RNA.…”

“…Construction of Zinc Finger Protein Expression Vectors. The construction of plasmid vectors used to express Xenopus TFIIIA and the Xenopus p43 zinc finger peptides p1-8, p1-4, and p6-9 have been described previously (33,35). The other zinc finger expressing cDNAs used in this study were constructed by the polymerase chain reaction (PCR) using upstream primers containing a recognition sequence for the restriction enzyme NcoI and downstream primers containing a recognition sequence for the restriction enzyme EcoRI.…”

Eukaryotic 5S rRNA biogenesis

“…Unlike TFIIIA, p43 does not bind directly to 5S rDNA 89. Zinc fingers 1–4 are essential for specific binding to 5S rRNA 90. Uncharacterized homologs of Xenopus P43 can be found in the genomes of Danio rerio (zebrafish) and Oryzias latipes (ricefish).…”

Photo-controlled metal-ion (Zn2+ and Cd2+) release in aqueous Tween-20 micelle solution

“…The light-activated release of metal ions has currently attracted considerable attention since metal photo-release could be a valuable tool for the delivery of biologically active metals, which are an essential cofactor in numerous enzymes that are critical for life, [1][2][3][4] or of heavy metal ions to study the mechanisms of metal-ion trafficking and applications, such as chemotherapy, where toxic metal ions could be released to induce cell death by catalyzing DNA and RNA cleavage or by binding active sites of protein competition with essential metals. [5][6][7][8] Zinc (Zn 2+ ) is the second most abundant transition element in biology and plays an important role in various fundamental biological processes, [9][10][11][12][13] such as gene transcription, regulation of metalloenzymes and neural signal transmission. Recent advances have shed light on the biological roles of zinc, particularly on its functions related to neurobiology.…”