Role of base G-2 of pre-tRNAfMet in cleavage site selection by Escherichia coli RNase P in vitro.
In this study, a protocol for the purification of fully active Escherichia coli RNase P holoenzyme from a strain overproducing both the C5 protein and the M1 RNA components is described. A total of 0. 8 mg of homogeneous enzyme, with a 1:1 protein/RNA subunit stoichiometry, was recovered from a 1 L bacterial culture. In addition, a convenient and reliable method based on capillary gel electrophoresis was developed to measure initial rates of pre-tRNA maturation by RNase P. Using these tools, the kinetic parameters of cleavage by RNase P of various mutants of pre-tRNAfMet showing maturation defects in vivo [Meinnel and Blanquet (1995) J. Biol. Chem. 270, 15906-15914] were investigated in vitro and the locations of cleavage sites were determined from the length of the various products of the reaction. The nucleotide at position -2 of pre-tRNAfMet is shown to be important only in the selection of the cleavage site, whereas it has no role in the efficiency of the reaction. It is concluded that base G-2 acts as an antideterminant by preventing an alternative cleavage by RNase P. In addition, the presence of G-2 alone is enough to fully compensate for the lack of a G at position +1 of pre-tRNAfMet.
Lazard
M
M
Laboratoire de Biochimie, Unité Mixte de Recherche No. 7654 du Centre National de la Recherche Scientifique, Palaiseau, France.
Meinnel
T
T
D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D003067:Coenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019075:Electrophoresis, Capillary; D004722:Endoribonucleases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D006147:Guanine / Q000502:physiology*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D012322:RNA Precursors / Q000235:genetics / Q000502:physiology*; D012323:RNA Processing, Post-Transcriptional; D016337:RNA, Catalytic / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012358:RNA, Transfer, Met / Q000235:genetics / Q000502:physiology*; D043262:Ribonuclease P
D003067:Coenzymes; D029968:Escherichia coli Proteins; D012322:RNA Precursors; D016337:RNA, Catalytic; D012358:RNA, Transfer, Met; C089238:fMet-tRNA(fMet); D006147:Guanine; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli