Abstract
We have used a collection of mutant forms of Xenopus transcription factor IIIA (TFIIIA) to study its interaction with 5S rRNA. This collection includes a set of nine mutant proteins, each of which contains a structural disruption in one of the nine zinc fingers of TFIIIA (broken-finger mutants), and a pair of complementary N- and C-terminal truncation mutants. Equilibrium and kinetic binding analyses in conjunction with RNAse protection and interference assays have been used to characterize the RNA-protein interaction in each case. We find that alternative binding modes are available for specific, high-affinity recognition of 5S rRNA by TFIIIA. These binding modes are distinct kinetically and structurally, and the mode of recognition adopted by wild-type TFIIIA when binding to intact 5S rRNA is dependent on the structural integrity of zinc fingers 5 and 6 in TFIIIA and continuity of the sugar-phosphate backbone in loop A of 5S rRNA. Disruption of any of these components allows adoption of one or more alternative modes of binding. In the wild-type TFIIIA-5S rRNA complex, some portions of TFIIIA, most notably the N-terminal three zinc fingers, are prevented from interacting with 5S rRNA in an energetically optimal way, and instead adopt a mode of binding that represents a compromise with the rest of the protein.
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