Identification of domains responsible for signal recognition and transduction within the QUTR transcription repressor protein.

QUTR (qutR-encoded transcription-repressing protein) is a multi-domain repressor protein active in the signal-transduction pathway that regulates transcription of the quinic acid utilization (qut) gene cluster in Aspergillus nidulans. In the presence of quinate, production of mRNA from the eight genes of the qut pathway is stimulated by the activator protein QUTA (qutA-encoded transcription-activating protein). Mutations in the qutR gene alter QUTR function such that the transcription of the qut gene cluster is permanently on (constitutive phenotype) or is insensitive to the presence of quinate (super-repressed phenotype). These mutant phenotypes imply that the QUTR protein plays a key role in signal recognition and transduction, and we have used deletion analysis to determine which regions of the QUTR protein are involved in these functions. We show that the QUTR protein recognizes and binds to the QUTA protein in vitro and that the N-terminal 88 amino acids of QUTR are sufficient to inactivate QUTA function in vivo. Deletion analysis and domain-swap experiments imply that the two C-terminal domains of QUTR are mainly involved in signal recognition.

Differential flux through the quinate and shikimate pathways. Implications for the channelling hypothesis.

The qutC gene encoding dehydroshikimate dehydratase has been constitutively overexpressed in Aspergillus nidulans from a range of 1-30-fold over the normal wild-type level. This overexpression leads to impaired growth in minimal medium which can be alleviated by the addition of aromatic amino acids to the medium. Overexpression of the qutC gene in mutant strains lacking protocatechuic acid (PCA) oxygenase leads to the build up of PCA in the medium, which can be measured by a simple assay. Measuring the rate of production of PCA in strains overproducing dehydroshikimate dehydratase and correlating this with the level of overproduction and impaired ability to grow in minimal medium lacking aromatic amino acids leads to the conclusion that (a) the metabolites 3-dehydroquinate and dehydroshikimate leak from the AROM protein at a rate comparable with the extent of flux catalysed by the AROM protein, (b) the AROM protein has a low-level channelling function probably as a result of the close juxtaposition of five active sites and (c) this channelling function is only physiologically significant under non-optimal conditions of nutrient supply and oxygenation, when the organism is in situ in its natural environment.