Role of RNA surveillance proteins Upf1/CpaR, Upf2 and Upf3 in the translational regulation of yeast CPA1 gene.

Gene CPA1, encoding one of the subunits of carbamoylphosphate synthetase (CPSase A) is subject to a translational control by arginine of which the essential element is a 25 amino acid peptide encoded by the CPA1 messenger. The peptide is the product of an open reading frame located upstream (uORF) of the coding phase of the gene, within a 250 nucleotide leader. In the past, a series of mutations impairing the repression of gene CPA1 by arginine had been selected in vivo.

The proper folding of a long C-terminal segment of the yeast Lys14p regulator is required for activation of LYS genes in response to the metabolic effector.

Transcription of lysine genes in Saccharomyces cerevisiae is dependent on Lys14p and on alpha-aminoadipate semialdehyde (alphaAASA), an intermediate of the pathway. The two-thirds C-terminal end of Lys14p is sufficient to ensure the activation function of the protein and its modulation by alphaAASA. Here, we show that no single discrete domain of Lys14p is able to activate transcription and that most of the deleted LexA-Lys14p proteins are inactive even in the presence of a high alphaAASA concentration.

Functional analysis of the leader peptide of the yeast gene CPA1 and heterologous regulation by other fungal peptides.

The 25-amino-acid leader peptide present at the 5' end of yeast CPA1 mRNA is responsible for the translational repression of that gene by arginine. We show here that the active domain of the yeast peptide is highly specific and extends over amino acids 6-23. The region between amino acids 6-21 is well conserved between similar peptides present upstream of CPA1-homologous genes in other fungi.

Characterisation of a tripartite nuclear localisation sequence in the regulatory protein Lys14 of Saccharomyces cerevisiae.

The Lys14 regulatory protein of Saccharomyces cerevisiae activates the expression of the LYS genes involved in the lysine biosynthetic pathway. Studies with a fused Lys14-green fluorescent protein reveal that Lys14p is localised to the nucleus, even under growth conditions leading to the absence of LYS gene expression. Lys14p nuclear localisation is mediated by a tripartite sequence made up of three short basic motifs located on the C-terminal side of the Zn cluster domain of Lys14p.

In Saccharomyces cerevisae, feedback inhibition of homocitrate synthase isoenzymes by lysine modulates the activation of LYS gene expression by Lys14p.

Expression of the structural genes for lysine biosynthesis responds to an induction mechanism mediated by the transcriptional activator Lys14p in the presence of alpha-aminoadipate semialdehyde (alphaAASA), an intermediate of the pathway acting as a coinducer. This activation is reduced by the presence of lysine in the growth medium, leading to apparent repression. In this report we demonstrate that Saccharomyces cerevisiae possesses two genes, LYS20 and LYS21, encoding two homocitrate synthase isoenzymes which are located in the nucleus.