Regulation of a Neurospora crassa extracellular RNase by phosphorus, nitrogen, and carbon derepressions

Abstract

A new extracellular RNase, designated N4, was detected in culture filtrates from Neurospora crassa and its regulation was studied. Limitation of a nutrient obtainable from RNA alone was not sufficient to cause enzyme derepression. The addition of RNA to the medium had no inductive effect, but the addition of exogenous protein caused enzyme production. With protein in the medium, N4 was derepressible for all three elemental nutrients obtainable from RNA: carbon, nitrogen, and phosphorus. Successful carbon derepression required the addition of a small amount of proteolytic activity to the cultures, as has been reported for the carbon-derepressible proteases of N. crassa. Exogenous protein affected RNase production before translation. Effects of the exogenous protein appeared similar to those previously reported for N. crassa protease induction. N4 was under the control of the nit-2 and nuc-1 gene products. nit-2 and nuc-1 mutants were unable to derepress enzyme synthesis for nitrogen and phosphorus limitation, respectively; however, these mutants responded like wild types to the other two states of derepression. Enzyme synthesis was constitutive in the preg mutant. Results indicate that the transcription of the N4 structural gene responds to multiple regulatory gene products from different regulatory circuits and that external protein affects the synthesis of classes of hydrolases other than proteases.

Characterization and comparison of a Neurospora crassa RNase purified from cultures undergoing each of three different states of derepression

“…We have found that RNase N4 is secreted from N. crassa mycelia under three different states of derepression (14). The enzyme probably functions by utilizing external RNA as a source of phosphorus, nitrogen, or carbon, since it is derepressed by limiting any of these three elements.…”

“…By employing the RNase assay described previously (14), we found only one polypeptide with RNase activity in cultures grown for the isolation of extracellular RNase. In each of the three purification procedures, only one peak of activity appeared in the elution profiles from the first chromatography; this activity remained a single peak in subsequent chromatograms.…”

“…RNase assay. The assay was performed as described previously (14). One unit of activity is defined as the amount of enzyme required to raise the non-acid-precipitable absorbance at 260 nm (A260) of yeast RNA 1.0 U in 10 min at 37°C.…”

“…Specificity toward homopolyribonucleotides was determined by reacting pure N4 with substrate (Sigma Chemical Co.) at 1 mg/ml (in 0.1 M Tris, 0.005 M EDTA [pH 7.5]). These preparations were sampled and precipitated, and A260 was determined as described in the RNase assay reported previously (14). A blank for each of the four homopolyribonucleotides that did not receive enzyme was subtracted from the respective experimental values.…”

“…RESULTS RNase N4 was purified from nitrogen-starved, phosphorus-starved, and carbon-starved cultures of N. crassa. Gelatin (0.25%) was used as the exogenous protein required for enzyme production in all three cases, and thermolysin was added to C-derepressed cultures as described previously (14). Purified N. crassa alkaline protease (15) was added to the culture filtrate from phosphorus-starved mycelia to digest the exogenous gelatin.…”