Substrate shape preference of Escherichia coli ribonuclease P ribozyme and holo enzyme using bottom-half part-shifting variants of pre-tRNA.
We showed previously that the bacterial ribonuclease P (RNase P) ribozyme has substrate shape preference depending on the concentrations of catalytically important magnesium ions. The ribozyme discriminates a canonical cloverleaf precursor tRNA from a hairpin RNA with a CCA-tag sequence at low concentrations of magnesium ions. By detailed analysis of the shape preference using the bottom-half part-shifting variants of a tRNA precursor, we showed that the RNAs in a T-shape structure can be substrates for the ribozyme reactions even at low concentrations of magnesium ions, and that the RNA in a natural L-shape is the best substrate for both the ribozyme and the holo enzyme. The results also showed that the position of the bottom-half part did not affect the cleavage site selection of a substrate by the enzyme. Our results are the first kinetic evidence to show the importance of the bottom-half part of tRNA molecule, and our result also showed that the holo enzyme can discriminate substrate shape as well as the ribozyme at low concentrations of metal ions.
Tanaka
Terumichi
T
Division of Bioscience and Biotechnology, Department of Ecological Engineering, Toyohashi University of Technology, Toyohashi, Aichi, Japan. tanakat@eco.tut.ac.jp
Nagai
Yasuhiro
Y
Kikuchi
Yo
Y
D029968:Escherichia coli Proteins / Q000378:metabolism*; D020035:Holoenzymes / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism*; D016337:RNA, Catalytic / Q000378:metabolism; D043262:Ribonuclease P / Q000378:metabolism*; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D020035:Holoenzymes; D012322:RNA Precursors; D016337:RNA, Catalytic; D043262:Ribonuclease P; D008274:Magnesium