Identification of a minimal domain of 5 S ribosomal RNA sufficient for high affinity interactions with the RNA-specific zinc fingers of transcription factor IIIA.
Transcription factor IIIA of Xenopuslaevis serves a dual function during oogenesis and early development: this zinc finger protein binds to the internal promoter element of the 5 S ribosomal RNA genes and acts as a positive transcription factor; additionally, the protein functions in 5 S RNA storage. The central four zinc fingers (zf4-7) of the nine-finger protein have been shown to bind 5 S rRNA with comparable or higher affinity than the full-length protein. The role of finger seven in binding affinity has been examined by deletion analysis. A zf4-6 protein binds 5 S RNA with about a sevenfold reduction in binding affinity, compared to zf4-7. The effect of non-specific competitor DNA on binding affinities of the zinc finger peptides was examined and found to have a significant effect on the measured affinities of these peptides for full-length and truncated versions of 5 S RNA. The interaction of zf4-6 with full-length 5 S RNA was far more sensitive to non-specific competitor concentration than was the zf4-7:5 S RNA interaction, suggesting that finger seven contributes to both affinity and specificity in this protein:RNA interaction. In order to map zinc finger binding sites on the 5 S RNA molecule, we generated truncated versions of the RNA and tested these molecules for their binding affinities with zf4-7 and zf4-6. Previous studies showed that a 75 nucleotide long RNA, comprising loop A, helix II, helix V, region E and helix IV, bound zf4-7 with high affinity. Selection and amplification binding assays (selex) have now been used to generate smaller high-affinity binding RNAs. We find that a 55 nucleotide long RNA, comprising loop A, helix V, region E and helix IV, but lacking helix II, retains high affinity for zf4-6. These data are consistent with the proposal that fingers 4-6 bind this central core of 5 S RNA and that finger seven binds the helix II region.
Neely
L S
LS
Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Lee
B M
BM
Xu
J
J
Wright
P E
PE
Gottesfeld
J M
JM
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005260:Female; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009865:Oocytes / Q000378:metabolism; D012341:RNA, Ribosomal, 5S / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis; D016335:Zinc Fingers / Q000235:genetics
D017931:DNA Primers; D004268:DNA-Binding Proteins; D012341:RNA, Ribosomal, 5S; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors