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

Abstract

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 decreasedk _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 increasedK _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.