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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tibs.2006.08.005 Byline: Alan G. Hinnebusch Abstract: Translation initiation in eukaryotes depends on many eukaryotic initiation factors (eIFs) that stimulate both recruitment of the initiator tRNA, Met-tRNA.sub.i.sup.Met, and mRNA to the 40S ribosomal subunit and subsequent scanning of the mRNA for the AUG start codon. The largest of these initiation factors, the eIF3 complex, organizes a web of interactions among several eIFs that assemble on the 40S subunit and participate in the different reactions involved in translation. Structural analysis suggests that eIF3 performs this scaffolding function by binding to the 40S subunit on its solvent-exposed surface rather than on its interface with the 60S subunit, where the decoding sites exist. This location of eIF3 seems ideally suited for its other proposed regulatory functions, including reinitiating translation on polycistronic mRNAs and acting as a receptor for protein kinases that control protein synthesis. Author Affiliation: Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA

The translation of General control noninducible 4 (GCN4) mRNA is derepressed in amino acid-deprived cells and it leads to transcriptional induction of nearly all genes encoding amino acid biosynthetic enzymes. The trans-acting proteins that control GCN4 translation have general functions in the initiation of protein synthesis or regulate the activities of initiation factors so that the molecular events that induce GCN4 translation also reduce the rate of general protein synthesis.

The eukaryotic 43S pre-initiation complex (PIC) containing Met-tRNAiMet in a ternary complex (TC) with eIF2-GTP scans the mRNA leader for an AUG codon in favorable "Kozak" context. AUG recognition triggers rearrangement of the PIC from an open conformation to a closed state with more tightly bound Met-tRNAiMet. Yeast ribosomal protein uS5/Rps2 is located at the mRNA entry channel of the 40S sub-unit in the vicinity of mRNA nucleotides downstream from the AUG codon or rRNA residues that communicate with the decoding center, but its participation in start codon recognition was unknown. We found that nonlethal substitutions of conserved Rps2 residues in the entry channel reduce bulk translation initiation and increase discrimination against poor initiation codons. A subset of these substitutions suppress initiation at near-cognate UUG start codons in a yeast mutant with elevated UUG initiation, and also increase discrimination against AUG codons in suboptimal Kozak context, thus resembling previously described substitutions in uS3/Rps3 at the 40S entry channel or initiation factors eIF1 and eIF1A. In contrast, other Rps2 substitutions selectively discriminate against either near-cognate UUG codons, or poor Kozak context of an AUG or UUG start codon. These findings suggest that different Rps2 residues are involved in distinct mechanisms involved in discriminating against different features of poor initiation sites in vivo. [ABSTRACT FROM AUTHOR]

Research has been conducted on the protein kinase GCN2 which is activated in amino acid-starved cells after binding the uncharged tRNA to the histidyl-tRNA synthase-related domain. Results indicate that protein kinase domain in GCN2 is defective.

A multifactor complex has been found to be an important translation initiation intermediate in vivo. Translation initiation factor 2 (eIF2) bound to GTP. transfers an initiator to the 40S ribosomal subunit. It has been shown that yeast eIF5 can bridge interaction in vitro between eIF3 and eIF2 by binding at the same time to the amino terminus of eIF3 subunit NIP1 and the amino-terminal half of eIF2(beta). This is dependent on a conserved two-part motif in the eIF5 carboxyl terminus.

Initiator methionyl tRNA maturation requires Gcd10p and Gcd14p, essential proteins that are subunits of a complex in which nuclear localization is prominent. Gcd10 mutants are defective in maturation of tRNA-Met. In addition, 1-methyladenosine formation in yeast tRNA requires Gcd10p. The need for 1-methyladenosine at position 58 may account for the role played by the Gcd10p-Gcd14p complex in the initiation phase of translation.

We report a fast, sensitive, and robust procedure for the identification of precise phosphorylation sites in proteins separated by polyacrylamide gel electrophoresis by a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF) and online capillary liquid chromatography electrospray tandem ion trap mass spectrometry (LC/ESI/MS/MS). With this procedure, a single phosphorylation site was identified on as little as 20 ng (500 fmol) of the baculovirusexpressed catalytic domain of myosin I heavy-chain kinase separated by gel electrophoresis. The phosphoprotein is digested in the gel with trypsin, and the resulting peptides are extracted with > 60% yield and analyzed by MALDI/TOF before and after digestion with a phosphatase to identify the phosphopeptides. The phosphopeptides are then separated and fragmented in an on-line LC/ESI ion trap mass spectrometer to identify the precise phosphorylation sites. This procedure eliminates any off-line HPLC separation and minimizes sample handling. The use of MALDI/TOF and LCQ, two types of mass spectrometers that are widely available to the biological community, will make this procedure readily accessible to biologists. We applied this technique to identify two autophosphorylation sites and to assign at least another 12 phosphorylation sites to two tryptic peptides in a series of experiments using a gel slice containing only 200 ng (3 pmol) of human double-stranded RNA-activated protein kinase expressed in a mutant strain of the yeast Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, expression of the transcriptional activator GCN4 increases at the translational level in response to starvation for an amino acid. The products of multiple GCD genes are required for efficient repression of GCN4 mRNA translation under nonstarvation conditions. The majority of the known GCD genes encode subunits of the general translation initiation factor eIF-2 or eIF-2B. To identify additional initiation factors in yeast, we characterized 65 spontaneously arising [Gcd.sup.-] mutants. In addition to the mutations that were complemented by known GCD genes or by GCN3, we isolated mutant alleles of two new genes named GCD14 and GCD15. Recessive mutations in these two genes led to highly unregulated GCN4 expression and to derepressed transcription of genes in the histidine biosynthetic pathway under GCN4 control. The derepression of GCN4 expression in gcd14 and gcd15 mutants occurred with little or no increase in GCN4 mRNA levels, and it was dependent on upstream open reading frames (uORFs) in GCN4 mRNA that regulate its translation. We conclude that GCD14 and GCD15 are required for repression of GCN4 mRNA translation by the uORFs under conditions of amino acid sufficiency. The gcd14 and gcd15 mutations confer a slow-growth phenotype on nutrient-rich medium, and gcd15 mutations are lethal when combined with a mutation in gcd13. Like other known GCD genes, GCD14 and GCD15 are therefore probably required for general translation initiation in addition to their roles in GCN4-specific translational control.

An eIF2B subcomplex of the GCN3, GCD7, and GCD2 subunits binds to eIF2 and has greater affinity for iEF2(alphaP), but has no nucleotide-exchange activity. GCD1 and GCD6 subunits form an eIF2B subcomplex that binds to eIF2 and eIF2(alphaP) equally. The latter subcomplex has greater nucleotide-exchange activity than wild-type eIF2B not held back by eIF2(alphaB). It seems that binding of eIF2(alphaB) to the regulatory subcomplex makes productive interaction impossible with the catalytic subcomplex. Independently, eIF2 binds distinct eIF2B subcomplexes that catalyze and regulate guanine-nucleotide exchange.

Peptide chain initiation in protein synthesis is regulated by eukaryotic initiation factor 2B (eIF-2B) in mammalian cells. A cloned cDNA of the alpha subunit of eIF-2B in rat comprises a polypeptide of 305 aa with a molecular mass of 33.7 kDa. The predicted amino acid sequence displays 42% similarity with that of the GCN3 protein of Saccharomyces cerevisiae.

Phosphorylation of translation initiation factor-2 (elF-2) is an adaptive mechanism for downregulating protein synthesis under conditions of starvation and stress. The yeast Saccharomyces has evolved a sophisticated means of increasing translation of GCN4 mRNA when elF-2 is phosphorylated, allowing the induction of an important stress-response protein when expression of most other genes is decreasing. Because translation of GCN4 mRNA is so tightly coupled to elF-2 activity, genetic analysis of this system has provided unexpected insights into the regulation of elF-2 and its guanine nucleotide exchange factor, elF-2B.

Mutations in the eukaryotic translation initiation factor 2B (eIF-2 alpha) structural gene that do not influence the rate of development of wild-type yeast but suppress the toxic influence of eIF-2 alpha hyperphosphorylation, catalyzed by mutationally stimulated versions of the protein kinase GCN2, were isolated. GCN2's phosphorylation of eIF-2 alpha in Saccharomyces cerevisiae causes the inhibition of general translation initiation and specific rise in translation of GCN4 mRNA. The interaction between eIF-2 and its recycling factor, eIF-2B, is changed by these mutations in a manner that reduces the inhibitory influence of phosphorylated eIF-2 on the important role of eIF-2B in translation initiation.

Abstract Background Eukaryotic translation initiation factor 4G (eIF4G) is thought to influence the translational efficiencies of cellular mRNAs by its roles in forming an eIF4F-mRNA-PABP mRNP that is competent for attachment of the 43S preinitiation complex, and in scanning through structured 5' UTR sequences. We have tested this hypothesis by determining the effects of genetically depleting eIF4G from yeast cells on global translational efficiencies (TEs), using gene expression microarrays to measure the abundance of mRNA in polysomes relative to total mRNA for ~5900 genes. Results Although depletion of eIF4G is lethal and reduces protein synthesis by ~75%, it had small effects (less than a factor of 1.5) on the relative TE of most genes. Within these limits, however, depleting eIF4G narrowed the range of translational efficiencies genome-wide, with mRNAs of better than average TE being translated relatively worse, and mRNAs with lower than average TE being translated relatively better. Surprisingly, the fraction of mRNAs most dependent on eIF4G display an average 5' UTR length at or below the mean for all yeast genes. Conclusions This finding suggests that eIF4G is more critical for ribosome attachment to mRNAs than for scanning long, structured 5' UTRs. Our results also indicate that eIF4G, and the closed-loop mRNP it assembles with the m7 G cap- and poly(A)-binding factors (eIF4E and PABP), is not essential for translation of most (if not all) mRNAs but enhances the differentiation of translational efficiencies genome-wide.