Evidence that binding of C5 protein to P RNA enhances ribozyme catalysis by influencing active site metal ion affinity.
The RNA subunit (P RNA) of the bacterial RNase P ribonucleoprotein is a ribozyme that catalyzes the Mg-dependent hydrolysis of pre-tRNA, but it requires an essential protein cofactor (P protein) in vivo that enhances substrate binding affinities and catalytic rates in a substrate dependent manner. Previous studies of Bacillus subtilis RNase P, containing a Type B RNA subunit, showed that its cognate protein subunit increases the affinity of metal ions important for catalysis, but the functional role of these ions is unknown. Here, we demonstrate that the Mg2+ dependence of the catalytic step for Escherichia coli RNase P, which contains a more common Type A RNA subunit, is also modulated by its cognate protein subunit (C5), indicating that this property is fundamental to P protein. To monitor specifically the binding of active site metal ions, we analyzed quantitatively the rescue by Cd2+ of an inhibitory Rp phosphorothioate modification at the pre-tRNA cleavage site. The results show that binding of C5 protein increases the apparent affinity of the rescuing Cd2+, providing evidence that C5 protein enhances metal ion affinity in the active site, and thus is likely to contribute significantly to rate enhancement at physiological metal ion concentrations.
Sun
Lei
L
Center for RNA Molecular Biology, Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
Harris
Michael E
ME
D001665:Binding Sites / Q000502:physiology; D002104:Cadmium / Q000378:metabolism; D002384:Catalysis; D002413:Cations, Divalent / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008670:Metals / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D021122:Protein Subunits / Q000378:metabolism*; D016337:RNA, Catalytic / Q000378:metabolism*; D043262:Ribonuclease P / Q000378:metabolism*
D002413:Cations, Divalent; D029968:Escherichia coli Proteins; D008670:Metals; D021122:Protein Subunits; D016337:RNA, Catalytic; D002104:Cadmium; D043262:Ribonuclease P; C470865:ribonuclease P, E coli; D008274:Magnesium