5 research outputs found

    Investigation of catalysis by bacterial RNase P via LNA and other modifications at the scissile phosphodiester

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    We analyzed cleavage of precursor tRNAs with an LNA, 2ā€²-OCH3, 2ā€²-H or 2ā€²-F modification at the canonical (c0) site by bacterial RNase P. We infer that the major function of the 2ā€²-substituent at nt āˆ’1 during substrate ground state binding is to accept an H-bond. Cleavage of the LNA substrate at the c0 site by Escherichia coli RNase P RNA demonstrated that the transition state for cleavage can in principle be achieved with a locked C3ā€² -endo ribose and without the H-bond donor function of the 2ā€²-substituent. LNA and 2ā€²-OCH3 suppressed processing at the major aberrant māˆ’1 site; instead, the m+1 (nt +1/+2) site was utilized. For the LNA variant, parallel pathways leading to cleavage at the c0 and m+1 sites had different pH profiles, with a higher Mg2+ requirement for c0 versus m+1 cleavage. The strong catalytic defect for LNA and 2ā€²-OCH3 supports a model where the extra methylene (LNA) or methyl group (2ā€²-OCH3) causes a steric interference with a nearby bound catalytic Mg2+ during its recoordination on the way to the transition state for cleavage. The presence of the protein cofactor suppressed the ground state binding defects, but not the catalytic defects

    Eukaryotic RNase P RNA mediates cleavage in the absence of protein

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    The universally conserved ribonucleoprotein RNase P is involved in the processing of tRNA precursor transcripts. RNase P consists of one RNA and, depending on its origin, a variable number of protein subunits. Catalytic activity of the RNA moiety so far has been demonstrated only for bacterial and some archaeal RNase P RNAs but not for their eukaryotic counterparts. Here, we show that RNase P RNAs from humans and the lower eukaryote Giardia lamblia mediate cleavage of four tRNA precursors and a model RNA hairpin loop substrate in the absence of protein. Compared with bacterial RNase P RNA, the rate of cleavage (kobs) was five to six orders of magnitude lower, whereas the affinity for the substrate (appKd) was reduced ā‰ˆ20- to 50-fold. We conclude that the RNA-based catalytic activity of RNase P has been preserved during evolution. This finding opens previously undescribed ways to study the role of the different proteins subunits of eukaryotic RNase P
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