Characterization of the spermidine-dependent, sequence-specific endoribonuclease that requires transfer RNA for its activity.
The spermidine-dependent, sequence-specific endoribonuclease (RNase 65) in mouse FM3A cells consists of protein and transfer RNA lacking its 3' terminus. In vitro properties of this enzyme were characterized using partially purified enzyme. The RNase 65 activity requires spermidine, which is not replaceable with spermine or Mg++. The enzyme cleaves an RNA substrate on the 3' side of the phosphodiester bond. The cleavage reaction has a temperature optimum around 50 degrees C and a pH optimum around 7.0. The optimum KCl concentration for the activity is around 10 mM. Relative cleavage efficiency of two differently folded RNA substrates with the common target sequence was analyzed at 37 degrees C and 50 degrees C. The results of this analysis suggest that unfolding of the target sequence is critical for recognition by RNase 65. Furthermore, in experiments using several point-mutated RNA substrates designed to form basically the same secondary structure as the wild type, one to three nucleotide substitutions in the target sequence all reduced cleavage efficiency. The RNase 65 activity is found only in cytosolic extracts, not in nuclear ones. Gel filtration analysis suggests that the native size of the endoribonuclease is approximately 150 kDa.
Nashimoto
M
M
Department of Biochemistry, Hokkaido University, School of Medicine, Sapporo, Japan.
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002850:Chromatography, Gel; D004722:Endoribonucleases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D012343:RNA, Transfer / Q000378:metabolism*; D013095:Spermidine / Q000378:metabolism*; D013096:Spermine / Q000378:metabolism; D013696:Temperature
D009838:Oligodeoxyribonucleotides; D013096:Spermine; D012343:RNA, Transfer; D004722:Endoribonucleases; C070374:ribonuclease 65; D008274:Magnesium; D013095:Spermidine