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J. Biol. Chem., Vol. 277, Issue 33, 30023-30030, August 16, 2002

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Mutations in the Rho Transcription Termination Factor That Affect RNA Tracking^*

Yi Xu, Harold Kohn^§, and William R. Widger^¶

From the  Department of Biology and Biochemistry, University of Houston, Houston, Texas 77204-5001 and the ^§ Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599-7360

Model studies have identified 16 conserved positively charged amino acids that form a positive strip pointing toward the center hole of Rho. Fourteen residues were individually changed to either an alanine or a glycine and one to a glutamate. Residues Arg²⁶⁹, Arg²⁷², Lys²⁸³, Arg²⁹⁶, Lys²⁹⁸, and Arg²⁹⁹ form a subdomain (locus) located N-terminal to (above) the ATP hydrolysis domain (P-loop) and mutations in these residues led to either inactive Rho or to proteins displaying decreased k_cat for poly(C)-dependent ATP hydrolysis, increased K_m for ribo(C)₁₀ activation, and decreased transcription termination efficiencies (57-77%) compared with wild-type Rho. Residues Arg³⁴⁷, Lys³⁴⁸, Lys³⁵², and Arg³⁵³ form a subdomain (locus) C-terminal to (below) the ATP hydrolysis domain, and mutations in these residues also show a decreased k_cat for poly(C)-dependent ATP hydrolysis, an increased K_m for ribo(C)₁₀ activation, and a 50-70% decrease in transcription termination, compared with wild-type Rho. Residues Arg²¹² and Lys³³⁶ surround the ATP hydrolysis domain, and mutations in these residues also altered the kinetic properties of Rho. We conclude that the secondary RNA-tracking site consists of amino acids whose putative orientation faces the central hole in Rho and in part reside in two clusters of positively charged residues located above and below the ATP hydrolysis domain.

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