The QUTA activator and QUTR repressor proteins of Aspergillus nidulans interact to regulate transcription of the quinate utilization pathway genes

Abstract

Genetic evidence suggests that the activity of the native QUTA transcription activator protein is negated by the action of the QUTR transcription repressor protein. When Aspergillus nidulans was transformed with plasmids containing the wild-type qutA gene, transformants that constitutively expressed the quinate pathway enzymes were isolated. The constitutive phenotype of these transformants was associated with an increased copy number of the transforming qutA gene and elevated qutA mRNA levels. Conversely, when A. nidulam was transformed with plasmids containing the qufR gene under the control of the constitutive pgk promoter, transformants with a superrepressed phenotype (unable to utilize quinate as a carbon source) were isolated. The super-repressed phenotype of these transformants was associated with an increased copy number of the transforming qutR gene and elevated qutR mRNA levels. These copy-number-dependent phenotypes argue that the levels of the QUTA and QUTR proteins were elevated in the high-copynumber transformants. When diploid strains were formed by combining haploid strains that contained high copy numbers of either the qutA gene (constitutive phenotype) or the qufR gene (super-repressing; non-inducible phenotype), the resulting diploid phenotype was one of quinate-inducible production of the quinate pathway enzymes, in a manner similar to wild-type. The simplest interpretation of these observations is that the QUTR repressor protein mediates its repressing activity through a direct interaction with the QUTA activator protein. Other possible interpretations are discussed in the text. Experiments in which truncated versions of the QUTA protein were produced in the presence of a wild-type QUTA protein indicate that the QUTR repressor protein recognizes and binds to the C-terminal half of the QUTA activator protein.

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

“…We have previously shown in A. nidulans that over-expression of the qutA gene produces a constitutive phenotype and that overexpression of the qutR gene produces a super-repressed phenotype in strains that are wild-type for the qut gene cluster [7]. When constitutive and super-repressed haploids are combined, the resulting diploid has an inducible phenotype, implying that the QUTA and QUTR proteins interact in i o [7].…”

“…The amino acid co-ordinates specified by the qutA and qutR sequences subcloned into the vectors pNUFC101, pNUFC102 [7], pET9d, pTrc99a and pSPUTK are shown in Table 1. The subcloning procedure for plasmids pTR108\113 and pNUFC285\315 is complex and requires explanation.…”

“…When constitutive and super-repressed haploids are combined, the resulting diploid has an inducible phenotype, implying that the QUTA and QUTR proteins interact in i o [7]. To test this implication directly and to determine which of the two domains of the activator QUTR binds to, we carried out coimmunoprecipitation studies in itro.…”

“…We chose to select for uracil-independent transformants using the pyr-4 gene of N. crassa, as this selection system produces transformants with a range of copy number of the transforming plasmid [7]. We chose to use the mutant strain 2035 as the host for transformation as it has a mutation in the qutR gene and has a recessive constitutive phenotype.…”

“…This activator is designated QA-1F in N. crassa and QUTA (qutA-encoded transcription-activating protein) in A. nidulans [1][2][3][4][5][6]. Experiments in i o have suggested that the QUTR protein mediates its repressing effect by binding to the QUTA protein [7]. The QUTR protein therefore has the complex job of both recognizing and binding to the QUTA protein and relatively small organic signal molecules.…”