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 Arg(269), Arg(272), Lys(283), Arg(296), Lys(298), and Arg(299) 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 Arg(347), Lys(348), Lys(352), and Arg(353) 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 Arg(212) and Lys(336) 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.
Highlights
Rho transcription termination factor is one of several nucleic acid-binding proteins belonging to a family of helicases with a homohexameric structure shaped like a toroid ring that utilizes the hydrolysis of ATP to move along the nucleic acid [1, 2]
The Rho monomer consists of 419 amino acid residues divided into two structural domains, a primary RNA-binding domain and the ATP hydrolysis domain, which is based upon structural similarity with F1-ATP synthase [12]
A model for Rho tracking coupled to ATP hydrolysis that relies on the structural similarity between Rho and the -subunit of F1-ATP synthase and utilizes residues facing inside of the central hole in Rho has been put forth [18]
Summary
A strip of positively charged amino acids on Rho positioned toward the inside of the hole was identified based on the structure of bovine F1-ATP synthase [12, 19] threaded with E. coli Rho sequence and energy minimized [18]. This model takes into account three active ATP hydrolysis sites per hexamer, sequential hydrolysis of ATP, Rho activation by short oligoribonucleotides, and the discrimination between hydrolytic and inactive subunits. We document the importance of positive charges for Rho to hydrolyze ATP, to bind RNA, and to terminate transcripts
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