Substrate shape specificity of E coli RNase P ribozyme is dependent on the concentration of magnesium ion.
The bacterial RNase P ribozyme can accept a hairpin RNA with CCA-3' tag sequence as well as a cloverleaf pre-tRNA as substrate in vitro, but the details are not known. By switching tRNA structure using an antisense guide DNA technique, we examined the Escherichia coli RNase P ribozyme specificity for substrate RNA of a given shape. Analysis of the RNase P reaction with various concentrations of magnesium ion revealed that the ribozyme cleaved only the cloverleaf RNA at below 10 mM magnesium ion. At 10 mM magnesium ion or more, the ribozyme also cleaved a hairpin RNA with a CCA-3' tag sequence. At above 20 mM magnesium ion, cleavage site wobbling by the enzyme in tRNA-derived hairpin occurred, and the substrate specificity of the enzyme became broader. Additional studies using another hairpin substrate demonstrated the same tendency. Our data strongly suggest that raising the concentration of metal ion induces a conformational change in the RNA enzyme.
Ando
Tomoaki
T
Department of Ecological Engineering, Toyohashi University of Technology, Tempaku-cho, Toyohashi, Aichi 441-8580.
Tanaka
Terumichi
T
Kikuchi
Yo
Y
D001483:Base Sequence; D002413:Cations, Divalent; D016373:DNA, Antisense / Q000032:analysis; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D008274:Magnesium / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D015347:RNA Probes; D012323:RNA Processing, Post-Transcriptional; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D043262:Ribonuclease P / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D002413:Cations, Divalent; D016373:DNA, Antisense; D029968:Escherichia coli Proteins; D012322:RNA Precursors; D015347:RNA Probes; D016337:RNA, Catalytic; D043262:Ribonuclease P; C470865:ribonuclease P, E coli; D008274:Magnesium