A segment of GCN4 mRNA containing the upstream AUG codons confers translational control upon a heterologous yeast transcript.

Abstract

GCN4 encodes a transcriptional activator in Saccharomyces cerevisiae that is regulated at the translational level. We show that an "240-nucleotide segment from the GCN4 mRNA leader containing four AUG codons is sufficient to confer translational control typical of GCN4 upon a GALIlacZ fusion transcript. Regulation of the hybrid transcript is dependent upon multiple positive (GCN) and negative (GCD) trans-acting factors shown to regulate GCN4 expression posttranscriptionally. This result limits the target sequences for these factors to a small internal segment of the GCN4 mRNA leader. The elimination of AUG codons within this segment substantially reduces the usual derepressing effect of mutations in five GCD genes upon GCN4-lacZ expression. This supports the idea that the products of these negative regulatory genes act by modulating the effects of the upstream AUG codons on translation of GCN4 mRNA.In Saccharomyces cerevisiae, transcription of a large number of unlinked genes encoding amino acid biosynthetic enzymes is stimulated by the GCN4 protein in response to starvation for any single amino acid (1-3). This response is attributable to increased expression of GCN4 in starvation conditions. Derepression of GCN4 is dependent upon the trans-acting positive effectors GCN2 and GCN3. Repression of GCN4 in nonstarvation conditions requires the products of multiple GCD genes (4, 5). Genetic evidence suggests that GCN2 and GCN3 act indirectly as positive effectors of GCN4 by negative regulation of GCD factors (1,(5)(6)(7).The leader of GCN4 mRNA contains four AUG codons, each of which initiates a short open reading frame of two or three codons. Removal of all four upstream AUG codons leads to constitutive derepression of GCN4 and bypasses the usual requirement for GCN2 and GCN3 for efficient GCN4 expression. A gcdl mutation leads to little additional derepression of GCN4 expression after removal of the upstream AUG codons. These findings suggest that GCN2, GCN3, and GCDJ regulate GCN4 by modulating the inhibitory effects of the upstream AUG codons on translation of GCN4 mRNA (4, 8-10). Four additional GCD genes are known to act as negative effectors of GCN4 expression (5), but their possible involvement in translational control of GCN4 has not been examined.The upstream AUG codons are located in an ="220-nucleotide internal segment of the "600-nucleotide GCN4 mRNA leader. We were interested in whether other sequences in the GCN4 transcript are required in conjunction with the upstream AUG codons for translational control. To address this possibility, we attempted to transfer the regulation characteristic of GCN4 to a heterologous yeast mRNA by inserting various segments from the GCN4 mRNA leader into the 5' untranslated region of a GALI-lacZ fusion transcript. Our results indicate that an -240-nucleotide leader segment containing the four AUG codons is sufficient to confer general amino acid control upon GALJ-lacZ mRNA. Expression of fusion enzyme from the hybrid transcript is regulated by GCN2, GCN3, and fiv...

The positive regulatory function of the 5'-proximal open reading frames in GCN4 mRNA can be mimicked by heterologous, short coding sequences.

“…The upstream AUG codons are required for translational control of GCN4 expression by the products of GCN2, GCN3, GCDJ, and GCDIO to GCD13. A -240-nt mRNA leader segment containing the four AUG codons is sufficient to confer regulation by these factors upon a heterologous yeast transcript (18,19). The data presented here suggest that two 30-to 40-bp segments containing URFs 1 and 4, respectively, are sufficient for a pattern of translational control typical of wild-type GCN4 mRNA.…”

“…By contrast, eliminating the AUG codons has no effect on the level of GCN4 mRNA. These data show that the upstream AUG codons mediate translational control of GCN4 expression by GCN and GCD regulatory factors (6,18,19,25).…”

“…Only when AUG codon 1 or 2 is present upstream from AUG codon 3 or 4 is there a strong requirement for GCD gene products to maintain repression of GCN4 expression in nonstarvation conditions. These findings led to the idea that recognition of AUG codons 1 and 2 suppresses the inhibitory effects of AUG codons 3 and 4 and that this interaction is the target of GCD negative regulatory function (18,19).…”

Roles of the negatively charged N-terminal extension of Saccharomyces cerevisiae ribosomal protein S5 revealed by characterization of a yeast strain containing human ribosomal protein S5

“…To investigate cap-dependent translation, we used a construct (p281), in which the lacZ gene is under control of the GAL1 promoter (Mueller et al 1987). LacZ expression in the mutant strain was reduced by z20%-25% compared to the WT strain (Fig.…”

“…The p281 plasmid containing lacZ under GAL1/10 promoter has been described previously (Mueller et al 1987). The p281-4-URE2 and p281-4-URE2_CTT vectors have also been previously described (Komar et al , 2005.…”