The Ribonuclease Activities of the Single-Strand-Specific Nucleases of Neurospora crassa

Abstract

A study of the ribonuclease activity of the single-strand-specific endonuclease of Neurospora crassa is reported. The enzyme degraded denatured, but not native, reovirus RNA. It had optimal activity with ribosomal RNA in the absence of added Mg2+ at pH 8.5 and was inhibited by ATP, mercaptoethanol, and EDTA. The inhibition by EDTA was overcome with Co2+ but not with Mg2+. The 5′-phosphoryl ends of the products of a partial digest comprised all four bases whereas 65% of the 3′-hydroxyl ends terminated with guanosine, indicating that the endonuclease had some base specificity. N. crassa endonuclease degraded purified yeast phenyl-alanine-specific tRNA (tRNAPhe) to large fragments with chain lengths of 13–14 nucleotides and to smaller fragments. This result indicated that the initial points of attack were the single-stranded "loop" regions of the tRNA molecule.Some new observations are also reported on the ribonuclease activity of single-strand-specific N. crassa exonuclease. In the presence of 10 mM Mg2+, the exonuclease removed the 3′-terminal AMP residue from tRNAPhe without "nicking" the molecule internally. When the concentration of Mg2+ was lowered to 1 mM, more extensive degradation of tRNA occurred.