Identification of a minimal domain of 5 S ribosomal RNA sufficient for high affinity interactions with the RNA-specific zinc fingers of transcriptionfactor IIIA

Transcription factor IIIA of Xenopus laevis serves a dual function during o ogenesis and early development: this zinc finger protein binds to the inter nal promoter element of the 5 S ribosomal RNA genes and acts as a positive transcription factor; additionally, the protein functions in 5 S RNA storag e. The central four zinc fingers (zf4-7) of the nine-finger protein have be en 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 exami ned by deletion analysis. A zf4-6 protein binds 5 S RNA with about a sevenf old reduction in binding affinity, compared to zf4-7. The effect of non-spe cific 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 in teraction of zf4-6 with full-length 5 S RNA was far more sensitive to non-s pecific competitor concentration than was the zf4-7:5 S RNA interaction, su ggesting 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 gen generated truncated versions of the RNA and te sted these molecules for their binding affinities with zf4-7 and zf4-6. Pre vious studies showed that a 75 nucleotide long RNA, comprising loop A, heli x ii, helix V, region E and helix IV, bound zf4-7 with high affinity. Selec tion and amplification binding assays (selex) have now been used to generat e smaller high-affinity binding RNAs. We find that a 55 nucleotide long RNA , comprising loop A, helix V, region E and helix TV, but lacking helix II, retains high affinity for zf4-6. These data are consistent with the proposa l that fingers 4-6 bind this central core of 5 S RNA and that finger seven binds the helix II region. (C) 1999 Academic Press.