Design and isolation of ribozyme-substrate pairs using RNase P-based ribozymes containing altered substrate binding sites.
Substrate recognition and cleavage by the bacterial RNase P RNA requires two domains, a specificity domain, or S-domain, and a catalytic domain, or C-domain. The S-domain binds the T stem-loop region in a pre-tRNA substrate to confer specificity for tRNA substrates. In this work, the entire S-domain of the Bacillus subtilis RNase P RNA is replaced with an artificial substrate binding module. New RNA substrates are isolated by in vitro selection using two libraries containing random regions of 60 nt. At the end of the selection, the cleavage rates of the substrate library are approximately 0.7 min(-1)in 10 mM MgCl(2)at 37 degrees C, approximately 4-fold better than the cleavage of a pre-tRNA substrate by the wild-type RNase P RNA under the same conditions. The contribution of the S-domain replacement to the catalytic efficiency is from 6- to 22 000-fold. Chemical and nuclease mapping of two ribozyme-product complexes shows that this contribution correlates with direct interactions between the S-domain replacement and the selected substrate. These results demonstrate the feasibility of design and isolation of RNase P-based, matching ribozyme-substrate pairs without prior knowledge of the sequence or structure of the interactive modules in the ribozyme or substrate.
Mobley
E M
EM
Department of Biochemistry and Molecular Biology, University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA.
Pan
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D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000235:genetics*; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism*; D012360:RNA, Transfer, Phe / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D043262:Ribonuclease P; D006163:Ribonuclease T1 / Q000378:metabolism; D013379:Substrate Specificity; D015003:Yeasts / Q000235:genetics
D012322:RNA Precursors; D016337:RNA, Catalytic; D012360:RNA, Transfer, Phe; C039919:RNA, recombinant; D012313:RNA; D012343:RNA, Transfer; D004722:Endoribonucleases; D043262:Ribonuclease P; C032825:ribonuclease V(1); D006163:Ribonuclease T1