Ernest M. Hannig

Complex formation by positive and negative translational regulators of GCN4.

GCN4 is a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae whose expression is regulated by amino-acid availability at the translational level. GCD1 and GCD2 are negative regulators required for the repression of GCN4 translation under nonstarvation conditions that is mediated by upstream open reading frames (uORFs) in the leader of GCN4 mRNA. GCD factors are thought to be antagonized by the positive regulators GCN1, GCN2 and GCN3 in amino acid-starved cells to allow for increased GCN4 protein synthesis. Previous genetic studies suggested that GCD1, GCD2, and GCN3 have closely related functions in the regulation of GCN4 expression that involve translation initiation factor 2 (eIF-2). In agreement with these predictions, we show that GCD1, GCD2, and GCN3 are integral components of a high-molecular-weight complex of approximately 600,000 Da. The three proteins copurified through several biochemical fractionation steps and could be coimmunoprecipitated by using antibodies against GCD1 or GCD2. Interestingly, a portion of the eIF-2 present in cell extracts also cofractionated and coimmunoprecipitated with these regulatory proteins but was dissociated from the GCD1/GCD2/GCN3 complex by 0.5 M KCl. Incubation of a temperature-sensitive gcdl-101 mutant at the restrictive temperature led to a rapid reduction in the average size and quantity of polysomes, plus an accumulation of inactive 80S ribosomal couples; in addition, excess amounts of eIF-2 alpha, GCD1, GCD2, and GCN3 were found comigrating with free 40S ribosomal subunits. These results suggest that GCD1 is required for an essential function involving eIF-2 at a late step in the translation initiation cycle. We propose that lowering the function of this high-molecular-weight complex, or of eIF-2 itself, in amino acid-starved cells leads to reduced ribosomal recognition of the uORFs and increased translation initiation at the GCN4 start codon. Our results provide new insights into how general initiation factors can be regulated to affect gene-specific translational control.

GCD11, a negative regulator of GCN4 expression, encodes the gamma subunit of eIF-2 in Saccharomyces cerevisiae.

The eukaryotic translation initiation factor eIF-2 plays a critical role in regulating the expression of the yeast transcriptional activator GCN4. Mutations in genes encoding the alpha and beta subunits of eIF-2 alter translational efficiency at the GCN4 AUG codon and constitutively elevate GCN4 translation. Mutations in the yeast GCD11 gene have been shown to confer a similar phenotype. The nucleotide sequence of the cloned GCD11 gene predicts a 527-amino-acid polypeptide that is similar to the prokaryotic translation elongation factor EF-Tu. Relative to EF-Tu, the deduced GCD11 amino acid sequence contains a 90-amino-acid N-terminal extension and an internal cysteine-rich sequence that contains a potential metal-binding finger motif. We have identified the GCD11 gene product as the gamma subunit of eIF-2 by the following criteria: (i) sequence identities with mammalian eIF-2 gamma peptides; (ii) increased eIF-2 activity in extracts prepared from cells cooverexpressing GCD11, eIF-2 alpha, and eIF-2 beta; and (iii) cross-reactivity of antibodies directed against the GCD11 protein with the 58-kDa polypeptide present in purified yeast eIF-2. The predicted GCD11 polypeptide contains all of the consensus elements known to be required for guanine nucleotide binding, suggesting that, in Saccharomyces cerevisiae, the gamma subunit of eIF-2 is responsible for GDP-GTP binding.

Eukaryotic Translation Initiation Factor 5 Is Critical for Integrity of the Scanning Preinitiation Complex and Accurate Control of GCN4 Translation

ABSTRACT The integrity of eukaryotic translation initiation factor (eIF) interactions in ribosomal preinitiation complexes is critical for the proper regulation of GCN4 mRNA translation in response to amino acid availability. Increased phosphorylation of eIF2 under amino acid starvation conditions leads to a corresponding increase in GCN4 mRNA translation. The carboxyl-terminal domain (CTD) of eIF5 (eIF5-CTD) has been identified as a potential nucleation site for preinitiation complex assembly. To further characterize eIF5 and delineate its role in GCN4 translational control, we isolated mutations leading to temperature sensitivity (Ts− phenotype) targeted at TIF5, the structural gene encoding eIF5 in yeast (Saccharomyces cerevisiae). Nine single point mutations were isolated, in addition to an allele in which the last 15 amino acids were deleted. The nine point mutations clustered in the eIF5-CTD, which contains two conserved aromatic/acidic boxes. Six of the point mutations derepressed GCN4 translation independent of eIF2 phosphorylation (Gcd− phenotype) at a permissive temperature, directly implicating eIF5-CTD in the eIF2/GTP/Met-tRNAiMet ternary complex binding process required for GCN4 translational control. In addition, stronger restriction of eIF5-CTD function at an elevated temperature led to failure to derepress GCN4 translation (Gcn− phenotype) in all of the mutants, most likely due to leaky scanning of the first upstream open reading frame of GCN4 mRNA. This latter result directly implicates eIF5-CTD in the process of accurate scanning for, or recognition of, AUG codons. Taken together, our results indicate that eIF5-CTD plays a critical role in both the assembly of the 43S complex and the postassembly process in the 48S complex, likely during the scanning process.

Ribosomal Protein L9 is the Product of GRC5, a Homolog of the Putative Tumor Suppressor QM in S. cerevisiae

Genes encoding members of the highly conserved QM family have been identified in eukaryotic organisms from yeast to man. Results of previous studies have suggested roles for QM in control of cell growth and proliferation, perhaps as a tumor suppressor, and in energy metabolism. We identified recessive lethal alleles of the Saccharomyces cerevisiae QM homolog GRC5 that increased GCN4 expression when present in multiple copies. These alleles encode truncated forms of the yeast QM protein Grc5p. Using a functional epitope‐tagged GRC5 allele, we localized Grc5p to a 60S fraction that contained the large ribosomal subunit. Two‐dimensional gel analysis of highly purified yeast ribosomes indicated that Grc5p corresponds to 60S ribosomal protein L9. This identification is consistent with the predicted physical characteristics of eukaryotic QM proteins, the highly biased codon usage of GRC5, and the presence of putative Rap1p‐binding sites in the 5′ sequences of the yeast GRC5 gene.

Analysis and reconstitution of translation initiation in vitro.

Publisher Summary This chapter discusses the analysis and reconstitution of translation initiation in vitro . Translation initiation is the rate-limiting step in protein biosynthesis, and alteration of the initiation factors by covalent modification—such as phosphorylation—or by mutation can have dramatic effects on the rate of protein synthesis. The budding yeast, Saccharomyces cerevisiae ( S.cerevisiae ), provides an ideal system to investigate structure–function relationships for conserved eukaryotic translation initiation factors (eIF) by combining powerful genetic tools with biochemical analysis of cell-free extracts and purified factors. Some of the individual reactions in the initiation pathway can be assayed in yeast whole cell extracts (WCEs) or by using the relevant purified factors in model assays. The chapter describes assays using both WCEs and purified elFs. For the latter, the chapter focuses on the formation of the ternary complex (TC), the recycling of eIF2-GDP to eIF2-GTP by eIF2, and stimulation of GTP hydrolysis in the TC by eIF5 The chapter describes the use of a single extract to assay the overall rate of protein synthesis with a luciferase reporter mRNA and the ability of the endogenous eIFs to deliver Met- Met i and mRNA to the 40S ribosome.