Nucleic Acids Research, 1992, Vol. 20, No. 14 3737-3742
© 1992
ENZYMOLOGY |
Characterization of the spermidine-dependent, sequence-specific endoribonuclease that rquires transfer RNA for its activity
Department of Biochemistry, Hokkaido University, School of Medicine Sapporo 060, Japan
Received March 6, 1992. Revised June 3, 1992. Accepted June 3, 1992.
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 spermidlne, which is not replaceable with spermine or Mg++. The enzyme cleaves an RNA substrate on the 3' side of the phosphodlester bond. The cleavage reaction has atemperature optimum around 50°C and a pH optimum around 7.0. The optimum KCI 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°C and 50°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 nucleotlde 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.
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