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J. Biol. Chem., Vol. 280, Issue 29, 26743-26750, July 22, 2005

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Mutations in TFIIIA That Increase Stability of the TFIIIA-5 S rRNA Gene Complex

UNUSUAL EFFECTS ON THE KINETICS OF COMPLEX ASSEMBLY AND DISSOCIATION^*

Kristina L. Brady, Stephen N. Ponnampalam, Michael J. Bumbulis, and David R. Setzer¶||

From the Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106 and the ^¶Division of Biological Sciences, University of Missouri, Columbia, Missouri 65211

We have identified four mutations in Xenopus TFIIIA that increase the stability of TFIIIA-5 S rRNA gene complexes. In each case, the mutation has a relatively modest effect on equilibrium binding affinity. In three cases, these equilibrium binding effects can be ascribed primarily to decreases in the rate constant for protein-DNA complex dissociation. In the fourth case, however, a substitution of phenylalanine for the wild-type leucine at position 148 in TFIIIA results in much larger compensating changes in the kinetics of complex assembly and dissociation. The data support a model in which a relatively unstable population of complexes with multi-component dissociation kinetics forms rapidly; complexes then undergo a slow conformational change that results in very stable, kinetically homogeneous TFIIIA-DNA complexes. The L148F mutant protein acts as a particularly potent transcriptional activator when it is fused to the VP16 activation domain and expressed in yeast cells. Substitution of L148 to tyrosine or tryptophan produces an equally strong transcriptional activator. Substitution to histidine results in genetic and biochemical effects that are more modest than, but similar to, those observed with the L148F mutation. We propose that an amino acid with a planar side chain at position 148 can intercalate between adjacent base pairs in the intermediate element of the 5 S rRNA gene. Intercalation occurs slowly but results in a very stable DNA-protein complex. These results suggest that transcriptional activation by a cis-acting sequence element is largely dependent on the kinetic, rather than the thermodynamic, stability of the complex formed with an activator protein. Thus, transcriptional activation is dependent in large part on the lifetime of the activator-DNA complex rather than on binding site occupancy at steady state. Introduction of intercalating amino acids into zinc finger proteins may be a useful tool for producing artificial transcription factors with particularly high in vivo activity.

Received for publication, March 10, 2005 , and in revised form, May 9, 2005.

^* This work was supported by National Institutes of Health Grant GM48035. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Department of Biology and Geology, Baldwin-Wallace College, Berea, OH 44017.

^|| To whom correspondence should be addressed: Division of Biological Sciences, 410 Tucker Hall, University of Missouri, Columbia, MO 65211. Tel.: 573-882-6821; Fax: 573-882-0123; E-mail: setzerd{at}missouri.edu.

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