The role of the central zinc fingers of transcription factor IIIA in binding to 5 S RNA.

In the nine-zinc finger Xenopus transcription factor TFIIIA the central group of fingers, fingers 4 to 7, have been shown to bind to 5 S RNA. In this study, we have attempted to assess the role of this region of the TFIIIA molecule in more detail than hitherto. High-resolution footprinting by RNases A and CV1 has been used to probe the binding to 5 S RNA of three TFIIIA peptides Tf(1-6), Tf(4-6) and Tf(4-7), consisting of fingers 1 to 6, 4 to 6, and 4 to 7, respectively, and of full-length TFIIIA. The results pinpoint the outer margins of binding of the central fingers within helices IV and II of TFIIIA. A comparison of the footprints reveals that the presence of finger 7 affords protection at positions C19 and U55, the distal portion of helix II and the proximal portion of loop B. In addition, our footprints suggest that the central fingers bind in the same manner, whether in an isolated group or in the intact TFIIIA molecule. In a companion study, we have determined the binding affinities of Tf(4-6) and Tf(4-7) for full-length and three truncated 5 S RNA molecules, the latter selected on the basis of the regions of the 5 S RNA molecule known to be important in the binding of TFIIIA. The analysis uses only fully active protein involved in the binding and the results are consistent with the corresponding footprinting results. This is the first time that a detailed study of the binding site of one particular zinc finger to RNA has been reported; the results should be of use in the design of 5 S RNA molecules and TFIIIA peptides for structural studies of the interaction between zinc fingers and RNA.

Searles MA ，Lu D ，Klug A 《JOURNAL OF MOLECULAR BIOLOGY》

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 LS ，Lee BM ，Xu J ，Wright PE ，Gottesfeld JM ... -  《JOURNAL OF MOLECULAR BIOLOGY》

Interaction of the RNA binding fingers of Xenopus transcription factor IIIA with specific regions of 5 S ribosomal RNA.

McBryant SJ ，Veldhoen N ，Gedulin B ，Leresche A ，Foster MP ，Wright PE ，Romaniuk PJ ，Gottesfeld JM ... -  《JOURNAL OF MOLECULAR BIOLOGY》

Dissection of the DNA-binding domain of Xenopus laevis TFIIIA. Quantitative DNase I footprinting analysis of specific complexes between a 5 S RNA gene fragment and N-terminal fragments of TFIIIA containing three, four or five zinc-finger domains.

Hansen PK ，Christensen JH ，Nyborg J ，Lillelund O ，Thøgersen HC ... -  《JOURNAL OF MOLECULAR BIOLOGY》

Studies on interaction of different zinc-finger domains of Xenopus laevis TFIIIA with eukaryotic 5S rRNAs.

Barciszewska MZ ，Giel-Pietraszuk M ，Thogersen HC ，Barciszewski J ... -  《-》