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 gro up 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 mo lecule 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 pe ptides 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 t he 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 fin ger 7 affords protection at positions C19 and U55, the distal portion of he lix II and the proximal portion of loop B. In addition, our footprints sugg est that the central fingers bind in the same manner, whether in an isolate d group or in the intact TFIIIA molecule. In a companion study, we have det ermined the binding affinities of Tf(4-6) and Tf(4-7) for full-length and t hree truncated 5 S RNA molecules, the latter selected on the basis of the r egions of the 5 S RNA molecule known to be important in the binding of TFII IA. The analysis uses only fully active protein involved in the binding and the results are consistent with the corresponding footprinting results. Th is is the first time that a detailed study of the binding site of one parti cular 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. (C) 2000 Academic Press.