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J. Biol. Chem., Vol. 279, Issue 18, 18370-18376, April 30, 2004

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Nucleotide Binding Induces Conformational Changes in Escherichia coli Transcription Termination Factor Rho^*

Yong-Joo Jeong, Dong-Eun Kim, and Smita S. Patel¶

From the Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854 and Department of Biotechnology and Bioengineering, Dong-Eui University, Gaya-dong 24, Busanjin-gu, Busan 614-714, Korea

The Escherichia coli Rho protein uses the energy of ATP binding and hydrolysis to translocate along RNA and cause transcription termination. Using fluorescence stopped-flow kinetic studies, we have discerned the conformational changes in the Rho protein that occur upon nucleotide and nucleic acid binding. We show that the 2', (3')-O-[N-methylanthraniloyl] derivative of ATP (mant-ATP) is a good fluorescent substrate of Rho and is hydrolyzed with a K_m comparable with that for ATP but a k_cat five to six times slower than that for ATP. The kinetics of ATP and mant-ATP binding indicates that, in the absence of RNA, the Rho protein is structurally distinct from the Rho hexamer found when bound to RNA or DNA. In the absence of RNA, the nucleotide-binding rates are 50- to 70-fold slower, and the dissociation rates are 40- to 120-fold slower than the corresponding rates in the presence of RNA. We conclude that RNA or DNA binding to the primary nucleic acid binding sites causes conformational changes in the Rho hexamer that result in the opening of the subunit interfaces. Furthermore, the kinetic studies revealed a unique protein conformational change in the Rho·RNA complex upon ATP binding that is a result of RNA contacting the secondary nucleic acid binding sites in the central channel of the Rho ring. This conformational change seems to render the Rho ring competent in ATP hydrolysis and translocation.

Received for publication, August 18, 2003 , and in revised form, February 16, 2004.

^* This work was supported by National Institutes of Health Grant 55310. 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.

^¶ To whom correspondence should be addressed. Tel.: 732-235-3372; Fax: 732-235-4783; E-mail: patelss{at}umdnj.edu.

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