Nucleic Acids Research, Vol 24, Issue 12 2377-2386, Copyright © 1996 by Oxford University Press
G Rotondo and D Frendewey
The pac1+ gene of the fission yeast Schizosaccharomyces pombe is essential
for viability and its overexpression induces sterility and suppresses
mutations in the pat1+ and snm1+ genes. The pac1+ gene encodes a protein
that is structurally similar to RNase III from Escherichia coli, but its
normal function is unknown. We report here the purification and
characterization of the Pac1 protein after overexpression in E. coli. The
purified protein is a highly active, double-strand-specific
endoribonuclease that converts long double- stranded RNAs into short
oligonucleotides and also cleaves a small hairpin RNA substrate. The Pac1
RNase is inhibited by a variety of double- and single-stranded
polynucleotides, but polycytidylic acid greatly enhances activity and also
promotes cleavage specificity. The Pac1 RNase produces 5'-phosphate termini
and requires Mg2+; Mn2+ supports activity but causes a loss of cleavage
specificity. Optimal activity was obtained at pH 8.5, at low ionic
strength, in the presence of a reducing agent. The enzyme is relatively
insensitive to N- ethylmaleimide but is strongly inhibited by ethidium
bromide and vanadyl ribonucleoside complexes. The properties of the Pac1
RNase support the hypothesis that it is a eukaryotic homolog of RNase III.
ARTICLES
Purification and characterization of the Pac1 ribonuclease of Schizosaccharomyces pombe
Department of Microbiology, New York University School of Medicine, NY 10016, USA.
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