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

Abstract

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.