Proof of de novo synthesis of the qa enzymes of Neurospora crassa during induction

Abstract

In Neurospora crassa three inducible enzymes are necessary to catabolize quinic acid to protocatechuic acid. The three genes encoding these enzymes are tightly linked on chromosome VII near methionine-7 (me-7). This qa cluster includes a fourth gene, qa-1, which encodes a regulatory protein apparently exerting positive control over transcription of the other three qa genes. However, an alternative hypothesis is that the qa-I protein simply activates preformed polypeptides derived from the three structural genes. The use of density labeling with D20 demonstrated conclusively that the qa enzymes are synthesized de novo only during induction on quinic acid. Native catabolic dehydroquinase (5-dehydroquinate dehydratase; 5-dehydroquinate hydro-lyase, EC 4.2.1.10) (a homopolymer of ca 22 identical subunits) has a density of 1.2790 g/cm3 as determined by centrifugation in a modified cesium chloride density gradient. Growth in H20 followed by induction in 95% D2O shifts the density of the enzyme to 1.3130 g/cm3, indicating de novo synthesis during induction. In the reciprocal experiment, i.e., growth in 80% D20 followed by induction in either 95% D20 or H20, the densities of catabolic dehydroquinase were 1.3135 and 1.2800 g/cm3, respectively. Because growth on D20 does not affect the density of the H20-induced enzyme, there can be no significant synthesis of catabolic dehydroquinase prior to induction. Similar results were obtained for a second qa enzyme, quinate dehydrogenase (quinate:NAD+ oxidoreductase, EC 1.1.1.24). Thus, induction of two qa enzymes involves de nQvo protein synthesis, not enzyme activation or assembly. In Neurospora crassa, the catabolism of quinic acid to acetate for use as a carbon source has been amply studied (1-3). Recent interest has centered on the initial three inducible catabolic enzymes necessary to convert quinic acid to protocatechuic acid (4-13). These enzymes are not physically associated with one another, yet the three genes encoding them are tightly linked on chromosome VII near me-7. In Giles, unpublished). This qa gene cluster also includes a fourth gene, qa-1, which apparently regulates the transcription of the three other genes in the cluster.There is strong genetic evidence that the qa-1 gene encodes a regulatory protein which, in the presence of quinic acid, exerts positive control over the synthesis of the enzymes encoded in the three adjacent structural genes (6, 10). The appearance of enzyme activity is very specifically regulated. In the presence of a preferred carbon source, e.g., sucrose, the activities of all three enzymes are quite low. Quinate as a sole carbon source causes a coordinate induction of all three enzymes (9). This increase in enzyme activity can be inhibited by the simultaneous addition of cycloheximide to the medium (ref. 5; J. A.Hautala, unpublished data; this paper). This effect strongly suggests that all three enzymes are being synthesized de novo during induction. An alternate explanation, which as yet has never been conclusively eliminate...

Genetic regulation of the qa gene cluster of Neurospora crassa: induction of qa messenger ribonucleic acid and dependency on qa-1 function.

“…The hypothesis was originally proposed, based largely on genetic evidence, that regulation of qa enzyme synthesis by the qa-1 + gene product, an activator protein, occurs at the level of transcription (10). This hypothesis is supported by evidence that induction of the qa enzymes involves de novo protein synthesis (20), indicating that regulation does not involve post-translational enzyme activation. More direct evidence for the transcriptional hypothesis comes from studies (V. B. Patel, C. Dykstra, and N. H. Giles In the present paper, additional evidence for transcriptional control of two qa genes (qa-3+ and qa-4+) is presented based on studies with the rabbit reticulocyte in vitro translation system (19).…”

“…Prior studies utilizing density labeling established that induction of the qa-2+ and qa-3+ gene products involves de novo protein synthesis (20 …”