A segment of mRNA encoding the leader peptide of the CPA1 gene confers repression by arginine on a heterologous yeast gene transcript.

The expression of the yeast gene CPA1, which encodes the small subunit of the arginine pathway carbamoylphosphate synthetase, is repressed by arginine at a translational level. CPA1 mRNA contains a 250-nucleotide-long leader which includes a 25-codon upstream open reading frame (uORF). Oligonucleotide site-directed mutagenesis of this uORF as well as sequencing of constitutive cis-dominant mutations has suggested that the leader peptide product of the CPA1 uORF is an essential negative element for repression of the CPA1 gene by arginine. In this work, a series of deletions affecting the regions 5' and 3' to the uORF in the leader sequence was constructed. The arginine-dependent repression of CPA1 was little affected in these constructions, indicating that these regions are not essential for the regulatory response. This conclusion was further supported by the finding that inserting the mRNA segment encoding the leader peptide sequence of CPA1 in the leader sequence of another gene, namely, GCN4, places this gene under arginine repression. Similarly, the behavior of fusions of the leader sequence of CPA1 with those of ARG4 or GAL10 confirmed that the regions of this leader located upstream and downstream from the uORF are dispensable for the regulation by arginine. Finally, a set of substitution mutations which modify the uORF nucleotide sequence while leaving unchanged the corresponding amino acid sequence was constructed. The mutations did not affect the repression of CPA1 by arginine. The data presented in this paper consequently agree with the conclusion that the leader peptide itself is the main element required for the translational repression of CPA1.     

Sequences 5' of the first upstream open reading frame in GCN4 mRNA are required for efficient translational reinitiation.

Translation of yeast GCN4 mRNA occurs by a reinitiation mechanism that is modulated by amino acid levels in the cell. Ribosomes which translate the first of four upstream open reading frames (uORFs) in the mRNA leader resume scanning and can reinitiate downstream. Under non-starvation conditions reinitiation occurs at one of the remaining three uORFs and GCN4 is repressed. Under starvation conditions, in contrast, ribosomes bypass the uORFs and reinitiate at GCN4 instead. The high frequency of reinitiation following uORF1 translation depends on an adequate distance to the next start codon and particular sequences surrounding the uORF1 stop codon. We present evidence that sequences 5' to uORF1 also strongly enhance reinitiation. First, reinitiation was severely inhibited when uORF1 was transplanted into the position of uORF4, even though the native sequence environment of the uORF1 stop codon was maintained, and this effect could not be accounted for by the decreased uORF1-GCN4 spacing. Second, insertions and deletions in the leader preceding uORF1 greatly reduced reinitiation at GCN4. Sequences 5' to uORF1 may influence the probability of ribosome release following peptide termination at uORF1. Alternatively, they may facilitate rebinding of an initiation factor required for reinitiation prior to resumption of the scanning process.     

cis-acting sequences involved in the translational control of GCN4 expression

Four short upstream open reading frames (uORFs) in the mRNA leader are required for the translational control of GCN4 expression in response to amino acid availability. Data are reviewed demonstrating that the fourth (3' proximal) uORF is sufficient to establish the repressed levels of GCN4 expression, while the first uORF functions as a positive regulatory element under starvation conditions to stimulate GCN4 translation. Furthermore, positive and negative trans-acting regulatory factors, the activities of which appear to be modulated according to amino acid availability, exert their effects on GCN4 expression through the uORFs. Direct comparison of the uORFs indicates that there are important nucleotide sequence differences between uORF1 and 4, and that these are located primarily around the termination codons of these elements. Recent findings suggest that the sequences that mediate repression of GCN4 expression are complex, but can be overcome under starvation conditions by ribosomes that have previously translated uORF1.     

The Evolutionarily Conserved Eukaryotic Arginine Attenuator Peptide Regulates the Movement of Ribosomes That Have Translated It

Translation of the upstream open reading frame (uORF) in the 5′ leader segment of the Neurospora crassa arg-2 mRNA causes reduced initiation at a downstream start codon when arginine is plentiful. Previous examination of this translational attenuation mechanism using a primer-extension inhibition (toeprint) assay in a homologous N. crassa cell-free translation system showed that arginine causes ribosomes to stall at the uORF termination codon. This stalling apparently regulates translation by preventing trailing scanning ribosomes from reaching the downstream start codon. Here we provide evidence that neither the distance between the uORF stop codon and the downstream initiation codon nor the nature of the stop codon used to terminate translation of the uORF-encoded arginine attenuator peptide (AAP) is important for regulation. Furthermore, translation of the AAP coding region regulates synthesis of the firefly luciferase polypeptide when it is fused directly at the N terminus of that polypeptide. In this case, the elongating ribosome stalls in response to Arg soon after it translates the AAP coding region. Regulation by this eukaryotic leader peptide thus appears to be exerted through a novel mechanism of cis-acting translational control.