Interplay between GCN2 and GCN4 expression, translation elongation factor 1 mutations and translational fidelity in yeast

Abstract

Genetic screens in Saccharomyces cerevisiae have identified the roles of ribosome components, tRNAs and translation factors in translational fidelity. These screens rely on the suppression of altered start codons, nonsense codons or frameshift mutations in genes involved in amino acid or nucleotide metabolism. Many of these genes are regulated by the General Amino Acid Control (GAAC) pathway. Upon amino acid starvation, the kinase GCN2 induces the GAAC cascade via increased translation of the transcriptional activator GCN4 controlled by upstream open reading frames (uORFs). Overexpression of the GCN2 or GCN4 genes enhances the sensitivity of translation fidelity assays that utilize genes regulated by GCN4, such as the suppression of a +1 insertion by S.cerevisiae translation elongation factor 1A (eEF1A) mutants. Paromomycin and the prion [PSI+], which reduce translational fidelity, do not increase GCN4 expression to induce the suppression phenotype and in fact reduce derepression. eEF1A mutations that reduce translation, however, reduce expression of GCN4 under non-starvation conditions. These eEF1A mutants also reduce HIS4 mRNA expression. Taken together, this system improves in vivo strategies for the analysis of translational fidelity and further provides new information on the interplay among translation fidelity, altered elongation and translational control via uORFs.

Elongation Factor 1A Is the Target of Growth Inhibition in Yeast Caused by Legionella pneumophila Glucosyltransferase Lgt1

“…Despite thorough and long lasting investigations, many aspects of mechanisms of its participation in protein synthesis and in other processes in eukaryotic cells remain unanswered. eEF1A has undergone thorough mutational analysis to discover amino acid residues important for its functioning in yeast (20,33,(35)(36)(37)(38). These studies were successful in identifying mutations that affected fidelity of translation, dependence upon nucleotide exchange factor, rate of GTP binding, and GTP hydrolysis.…”

Near-Cognate Codons Contribute Complexity to Translation Regulation

“…Homologs of GCN4 in other fungi contain at least two uORFs believed to have similar regulatory functions as the S. cerevisiae GCN4 uORFs. In addition to these regulatory uORFs, kinases such as Gcn2 also play a regulatory role in GCN4 expression; phosphorylation of the translation initiation factor eIF2α (α subunit of eukaryotic initiation factor 2) by Gcn2 kinase increases reinitiation at the GCN4 protein coding start codon in response to amino acid limitation ( 11 ).…”

Inbuilt Tendency of the eIF2 Regulatory System to Counteract Uncertainties

“…The phosphorylation process is one of the main disruptors of translation activity, converting eIF2 to a dominant inhibitor of the guanine nucleotide exchange factor (GEF) eIF2B, which disrupts the upcoming round of translation activity. The phosphorylated eIF2 loses the affinity with eIF2B or converts from a substrate to an inhibitor of eIF2B, and hence is unable to re-energise its active state with the help of eIF2B, resulting in disruption of the translation process [11], [12].…”