Claudio Scazzocchio — Page 2

PubMed2002

PrnA, a Zn2Cys6 activator with a unique DNA recognition mode, requires inducer for in vivo binding.

The PrnA transcriptional activator of Aspergillus nidulans binds as a dimer to CCGG-N-CCGG inverted repeats and to CCGG-6/7N-CCGG direct repeats. The binding specificity of the PrnA Zn cluster differs from that of the Gal4p/Ppr1p/UaY/Put3p group of proteins. Chimeras with UaY, a protein that strictly recognizes a CGG-6N-CCG motif, show that the recognition of the direct repeats necessitates the PrnA dimerization and linker elements, but the recognition of the CCGG-N-CCGG inverted repeats depends crucially on the PrnA Zn binuclear cluster and/or on residues amino-terminal to it. Three high-affinity sites in two different promoters have been visualized by in vivo methylation protection. Proline induction is essential for in vivo binding to these three sites but, as shown previously, not for nuclear entry. Simultaneous repression by ammonium and glucose does not affect in vivo binding to these high-affinity sites. PrnA differs from the isofunctional Saccharomyces cerevisiae protein Put3p, both in its unique binding specificity and in the requirement of induction for in vivo DNA binding.

Aspergillus niger↗

PubMed2002

The absence of molybdenum cofactor sulfuration is the primary cause of the flacca phenotype in tomato plants.

The molybdenum cofactor (MoCo)-containing enzymes aldehyde oxidase (AO; EC 1.2.3.1) and xanthine dehydrogenase (XDH; EC 1.2.1.37) require for activity a sulfuration step that inserts a terminal sulfur ligand into the MoCo. The tomato flacca mutation was originally isolated as a wilty phenotype due to a lack of abscisic acid (ABA) that is related to simultaneous loss of AO and XDH activities. An expressed sequence tag candidate from tomato was selected on the basis of homology to sulfurases from animals, fungi and the recently isolated Arabidopsis genes LOS5/ABA3. The tomato homologue maps as a single gene to the bottom of chromosome 7, consistent with the genetic location of the flacca mutation. The structure of FLACCA shows a multidomain protein with an N-terminal NifS-like sulfurase domain; a mammal-specific intermediate section; and a C-terminus containing conserved motifs. Prominent among these are molybdopterin oxidoreductases and thioredoxin redox-active centre/iron-sulfur-binding region signatures which may be relevant to the specific sulfuration of MoCo. Indeed, the molecular analysis of flacca identifies the mutation in a highly conserved motif located in the C-terminus. Activity gel assays show that FLACCA is expressed throughout the plant. Transient and stable complementation of flacca and the Arabidopsis aba3 mutants with Aspergillus nidulans hxB and FLACCA yielded full, partial and tissue-specific types of Mo-hydroxylase activities. Restoration of activity in the root alone is sufficient to augment plant ABA content and rectify the wild-type phenotype. Thus the pleiotropic flacca phenotype is due to the loss of activity of enzymes requiring a sulfurated MoCo.

Abscisic Acid↗