Breaking the Silos of Protein Synthesis.

Protein synthesis requires factors that are proposed to enhance discrete steps. Eukaryotic initiation factor eIF5A was initially thought to affect initiation; however, it was later shown to facilitate translation elongation at polyproline. Recent work by Schuller et al. demonstrates that eIF5A facilitates both general elongation and termination in yeast, challenging these steps as silos.

Mutations in the chromodomain-like insertion of translation elongation factor 3 compromise protein synthesis through reduced ATPase activity.

Translation elongation is mediated by ribosomes and multiple soluble factors, many of which are conserved across bacteria and eukaryotes. During elongation, eukaryotic elongation factor 1A (eEF1A; EF-Tu in bacteria) delivers aminoacylated-tRNA to the A-site of the ribosome, whereas eEF2 (EF-G in bacteria) translocates the ribosome along the mRNA. Fungal translation elongation is striking in its absolute requirement for a third factor, the ATPase eEF3. eEF3 binds close to the E-site of the ribosome and has been proposed to facilitate the removal of deacylated tRNA from the E-site. eEF3 has two ATP binding cassette (ABC) domains, the second of which carries a unique chromodomain-like insertion hypothesized to play a significant role in its binding to the ribosome. This model was tested in the current study using a mutational analysis of the Sac7d region of the chromodomain-like insertion. Specific mutations in this domain result in reduced growth rate as well as slower translation elongation. In vitro analysis demonstrates that these mutations do not affect the ability of eEF3 to interact with the ribosome. Kinetic analysis revealed a larger turnover number for ribosomes in comparison to eEF3, indicating that the partial reactions involving the ribosome are significantly faster than that of eEF3. Mutations in the chromodomain-like insertion severely compromise the ribosome stimulated ATPase of eEF3, strongly suggesting that it exerts an allosteric effect on the hydrolytic activity of eEF3. The chromodomain-like insertion is, therefore, vital to eEF3 function and may be targeted for developing novel antifungal drugs.

Translation elongation factor 1A mutants with altered actin bundling activity show reduced aminoacyl-tRNA binding and alter initiation via eIF2α phosphorylation.

Apart from its canonical function in translation elongation, eukaryotic translation elongation factor 1A (eEF1A) has been shown to interact with the actin cytoskeleton. Amino acid substitutions in eEF1A that reduce its ability to bind and bundle actin in vitro cause improper actin organization in vivo and reduce total translation. Initial in vivo analysis indicated the reduced translation was through initiation. The mutant strains exhibit increased levels of phosphorylated initiation factor 2α (eIF2α) dependent on the presence of the general control non-derepressible 2 (Gcn2p) protein kinase. Gcn2p causes downregulation of total protein synthesis at initiation in response to increases in deacylated tRNA levels in the cell. Increased levels of eIF2α phosphorylation are not due to a general reduction in translation elongation as eEF2 and eEF3 mutants do not exhibit this effect. Deletion of GCN2 from the eEF1A actin bundling mutant strains revealed a second defect in translation. The eEF1A actin-bundling proteins exhibit changes in their elongation activity at the level of aminoacyl-tRNA binding in vitro. These findings implicate eEF1A in a feedback mechanism for regulating translation at initiation.

ADP-ribosylation of translation elongation factor 2 by diphtheria toxin in yeast inhibits translation and cell separation.

Eukaryotic translation elongation factor 2 (eEF2) facilitates the movement of the peptidyl tRNA-mRNA complex from the A site of the ribosome to the P site during protein synthesis. ADP-ribosylation (ADP(R)) of eEF2 by bacterial toxins on a unique diphthamide residue inhibits its translocation activity, but the mechanism is unclear. We have employed a hormone-inducible diphtheria toxin (DT) expression system in Saccharomyces cerevisiae which allows for the rapid induction of ADP(R)-eEF2 to examine the effects of DT in vivo. ADP(R) of eEF2 resulted in a decrease in total protein synthesis consistent with a defect in translation elongation. Association of eEF2 with polyribosomes, however, was unchanged upon expression of DT. Upon prolonged exposure to DT, cells with an abnormal morphology and increased DNA content accumulated. This observation was specific to DT expression and was not observed when translation elongation was inhibited by other methods. Examination of these cells by electron microscopy indicated a defect in cell separation following mitosis. These results suggest that expression of proteins late in the cell cycle is particularly sensitive to inhibition by ADP(R)-eEF2.

Synergistic roles of eukaryotic translation elongation factors 1Bγ and 1A in stimulation of tombusvirus minus-strand synthesis.

Host factors are recruited into viral replicase complexes to aid replication of plus-strand RNA viruses. In this paper, we show that deletion of eukaryotic translation elongation factor 1Bgamma (eEF1Bγ) reduces Tomato bushy stunt virus (TBSV) replication in yeast host. Also, knock down of eEF1Bγ level in plant host decreases TBSV accumulation. eEF1Bγ binds to the viral RNA and is one of the resident host proteins in the tombusvirus replicase complex. Additional in vitro assays with whole cell extracts prepared from yeast strains lacking eEF1Bγ demonstrated its role in minus-strand synthesis by opening of the structured 3' end of the viral RNA and reducing the possibility of re-utilization of (+)-strand templates for repeated (-)-strand synthesis within the replicase. We also show that eEF1Bγ plays a synergistic role with eukaryotic translation elongation factor 1A in tombusvirus replication, possibly via stimulation of the proper positioning of the viral RNA-dependent RNA polymerase over the promoter region in the viral RNA template.These roles for translation factors during TBSV replication are separate from their canonical roles in host and viral protein translation.

Evidence that eukaryotic translation elongation factor 1A (eEF1A) binds the Gcn2 protein C terminus and inhibits Gcn2 activity.

The eukaryotic elongation factor 1A (eEF1A) delivers aminoacyl-tRNAs to the ribosomal A-site during protein synthesis. To ensure a continuous supply of amino acids, cells harbor the kinase Gcn2 and its effector protein Gcn1. The ultimate signal for amino acid shortage is uncharged tRNAs. We have proposed a model for sensing starvation, in which Gcn1 and Gcn2 are tethered to the ribosome, and Gcn1 is directly involved in delivering uncharged tRNAs from the A-site to Gcn2 for its subsequent activation. Gcn1 and Gcn2 are large proteins, and these proteins as well as eEF1A access the A-site, leading us to investigate whether there is a functional or physical link between these proteins. Using Saccharomyces cerevisiae cells expressing His(6)-eEF1A and affinity purification, we found that eEF1A co-eluted with Gcn2. Furthermore, Gcn2 co-immunoprecipitated with eEF1A, suggesting that they reside in the same complex. The purified GST-tagged Gcn2 C-terminal domain (CTD) was sufficient for precipitating eEF1A from whole cell extracts generated from gcn2Δ cells, independently of ribosomes. Purified GST-Gcn2-CTD and purified His(6)-eEF1A interacted with each other, and this was largely independent of the Lys residues in Gcn2-CTD known to be required for tRNA binding and ribosome association. Interestingly, Gcn2-eEF1A interaction was diminished in amino acid-starved cells and by uncharged tRNAs in vitro, suggesting that eEF1A functions as a Gcn2 inhibitor. Consistent with this possibility, purified eEF1A reduced the ability of Gcn2 to phosphorylate its substrate, eIF2α, but did not diminish Gcn2 autophosphorylation. These findings implicate eEF1A in the intricate regulation of Gcn2 and amino acid homeostasis.

Translation elongation factor 1A facilitates the assembly of the tombusvirus replicase and stimulates minus-strand synthesis.

Replication of plus-strand RNA viruses depends on host factors that are recruited into viral replicase complexes. Previous studies showed that eukaryotic translation elongation factor (eEF1A) is one of the resident host proteins in the highly purified tombusvirus replicase complex. Using a random library of eEF1A mutants, we identified one mutant that decreased and three mutants that increased Tomato bushy stunt virus (TBSV) replication in a yeast model host. Additional in vitro assays with whole cell extracts prepared from yeast strains expressing the eEF1A mutants demonstrated several functions for eEF1A in TBSV replication: facilitating the recruitment of the viral RNA template into the replicase complex; the assembly of the viral replicase complex; and enhancement of the minus-strand synthesis by promoting the initiation step. These roles for eEF1A are separate from its canonical role in host and viral protein translation, emphasizing critical functions for this abundant cellular protein during TBSV replication.

Structure of eEF3 and the mechanism of transfer RNA release from the E-site.

Elongation factor eEF3 is an ATPase that, in addition to the two canonical factors eEF1A and eEF2, serves an essential function in the translation cycle of fungi. eEF3 is required for the binding of the aminoacyl-tRNA-eEF1A-GTP ternary complex to the ribosomal A-site and has been suggested to facilitate the clearance of deacyl-tRNA from the E-site. Here we present the crystal structure of Saccharomyces cerevisiae eEF3, showing that it consists of an amino-terminal HEAT repeat domain, followed by a four-helix bundle and two ABC-type ATPase domains, with a chromodomain inserted in ABC2. Moreover, we present the cryo-electron microscopy structure of the ATP-bound form of eEF3 in complex with the post-translocational-state 80S ribosome from yeast. eEF3 uses an entirely new factor binding site near the ribosomal E-site, with the chromodomain likely to stabilize the ribosomal L1 stalk in an open conformation, thus allowing tRNA release.

The eukaryotic translation elongation Factor 1Bgamma has a non-guanine nucleotide exchange factor role in protein metabolism.

The turnover of damaged proteins is critical to cell survival during stressful conditions such as heat shock or oxidative stress. The accumulation of misfolded proteins in the endoplasmic reticulum (ER) is toxic to cells. Therefore these proteins must be efficiently exported from the ER and degraded by the proteasome or the vacuole. Previously it was shown that the loss of eukaryotic elongation factor 1Bγ (eEF1Bγ) from the yeast Saccharomyces cerevisiae results in resistance to oxidative stress. Strains lacking eEF1Bγ show severe defects in protein turnover during conditions of oxidative stress. Furthermore, these strains accumulate a greater amount of oxidized proteins, which correlates with changes in heat shock chaperones. These strains show severe defects in vacuole morphology and defects related to the maturation of carboxypeptidase Y that is not dependent on the catalytic subunit of the eEF1B complex as a guanine nucleotide exchange factor. Finally, eEF1Bγ co-immunoprecipitates with an essential component of ER-Golgi transport vesicles. Taken together, these results support a broader protein metabolism role for eEF1Bγ.

eEF1A: thinking outside the ribosome.

Eukaryotic translation elongation factor 1A (eEF1A) is one of the most abundant protein synthesis factors. eEF1A is responsible for the delivery of all aminoacyl-tRNAs to the ribosome, aside from initiator and selenocysteine tRNAs. In addition to its roles in polypeptide chain elongation, unique cellular and viral activities have been attributed to eEF1A in eukaryotes from yeast to plants and mammals. From preliminary, speculative associations to well characterized biochemical and biological interactions, it is clear that eEF1A, of all the translation factors, has been ascribed the most functions outside of protein synthesis. A mechanistic understanding of these non-canonical functions of eEF1A will shed light on many important biological questions, including viral-host interaction, subcellular organization, and the integration of key cellular pathways.

Targeting of eEF1A with Amaryllidaceae isocarbostyrils as a strategy to combat melanomas.

Melanomas display poor response rates to adjuvant therapies because of their intrinsic resistance to proapoptotic stimuli. This study indicates that such resistance can be overcome, at least partly, through the targeting of eEF1A elongation factor with narciclasine, an Amaryllidaceae isocarbostyril controlling plant growth. Narciclasine displays IC(50) growth inhibitory values between 30-100 nM in melanoma cell lines, irrespective of their levels of resistance to proapoptotic stimuli. Normal noncancerous cell lines are much less affected. At nontoxic doses, narciclasine also significantly improves (P=0.004) the survival of mice bearing metastatic apoptosis-resistant melanoma xenografts in their brain. The eEF1A targeting with narciclasine (50 nM) leads to 1) marked actin cytoskeleton disorganization, resulting in cytokinesis impairment, and 2) protein synthesis impairment (elongation and initiation steps), whereas apoptosis is induced at higher doses only (≥200 nM). In addition to molecular docking validation and identification of potential binding sites, we biochemically confirmed that narciclasine directly binds to human recombinant and yeast-purified eEF1A in a nanomolar range, but not to actin or elongation factor 2, and that 5 nM narciclasine is sufficient to impair eEF1A-related actin bundling activity. eEF1A is thus a potential target to combat melanomas regardless of their apoptosis-sensitivity, and this finding reconciles the pleiotropic cytostatic of narciclasine. -

Genome-wide screen of Saccharomyces cerevisiae null allele strains identifies genes involved in selenomethionine resistance.

Selenomethionine (SeMet) is a potentially toxic amino acid, and yet it is a valuable tool in the preparation of labeled proteins for multiwavelength anomalous dispersion or single-wavelength anomalous dispersion phasing in X-ray crystallography. The mechanism by which high levels of SeMet exhibits its toxic effects in eukaryotic cells is not fully understood. Attempts to use Saccharomyces cerevisiae for the preparation of fully substituted SeMet proteins for X-ray crystallography have been limited. A screen of the viable S. cerevisiae haploid null allele strain collection for resistance to SeMet was performed. Deletion of the CYS3 gene encoding cystathionine gamma-lyase resulted in the highest resistance to SeMet. In addition, deletion of SSN2 resulted in both increased resistance to SeMet as well as reduced levels of Cys3p. A methionine auxotrophic strain lacking CYS3 was able to grow in media with SeMet as the only source of Met, achieving essentially 100% occupancy in total proteins. The CYS3 deletion strain provides advantages for an easy and cost-effective method to prepare SeMet-substituted protein in yeast and perhaps other eukaryotic systems.

Mutational analysis reveals potential phosphorylation sites in eukaryotic elongation factor 1A that are important for its activity.

Previous studies have suggested that phosphorylation of translation elongation factor 1A (eEF1A) can alter its function, and large-scale phospho-proteomic analyses in Saccharomyces cerevisiae have identified 14 eEF1A residues phosphorylated under various conditions. Here, a series of eEF1A mutations at these proposed sites were created and the effects on eEF1A activity were analyzed. The eEF1A-S53D and eEF1A-T430D phosphomimetic mutant strains were inviable, while corresponding alanine mutants survived but displayed defects in growth and protein synthesis. The activity of an eEF1A-S289D mutant was significantly reduced in the absence of the guanine nucleotide exchange factor eEF1Bα and could be restored by an exchange-deficient form of the protein, suggesting that eEF1Bα promotes eEF1A activity by a mechanism other than nucleotide exchange. Our data show that several of the phosphorylation sites identified by high-throughput analysis are critical for eEF1A function.

Improper organization of the actin cytoskeleton affects protein synthesis at initiation.

Although the actin cytoskeleton and the translation machinery are considered to be separate cellular complexes, growing evidence supports overlapping regulation of the two systems. Because of its interaction with actin, the eukaryotic translation elongation factor 1A (eEF1A) is proposed to be a regulator or link between these processes. Using a genetic approach with the yeast Saccharomyces cerevisiae, specific regions of eEF1A responsible for actin interactions and bundling were identified. Five new mutations were identified along one face of eEF1A. Dramatic changes in cell growth, cell morphology, and actin cable and patch formation as well as a unique effect on total translation in strains expressing the F308L or S405P eEF1A mutant form were observed. The translation effects do not correlate with reduced translation elongation but instead include an initiation defect. Biochemical analysis of the eEF1A mutant forms demonstrated reduced actin-bundling activity in vitro. Reduced total translation and/or the accumulation of 80S ribosomes in strains with either a mutation or a null allele of genes encoding actin itself or actin-regulating proteins Tpm1p, Mdm20p, and Bnirp/Bni1p was observed. Our data demonstrate that eEF1A, other actin binding proteins, and actin mutants affect translation initiation through the actin cytoskeleton.

Translation elongation factor 1A is essential for regulation of the actin cytoskeleton and cell morphology.

The binding of eukaryotic translation elongation factor 1A (eEF1A) to actin is a noncanonical function that may link two distinct cellular processes, cytoskeleton organization and gene expression. Using the yeast Saccharomyces cerevisiae, we have established an in vivo assay that directly identifies specific regions and residues of eEF1A responsible for actin interactions and bundling. Using a unique genetic screen, we isolated a series of eEF1A mutants with reduced actin bundling activity. These mutations alter actin cytoskeleton organization but not translation, indicating that these are separate functions of eEF1A. This demonstrates for the first time a direct consequence of eEF1A on cytoskeletal organization in vivo and the physiological significance of this interaction.

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.

Ribosomal protein (rp) S5 belongs to a family of ribosomal proteins that includes bacterial rpS7. rpS5 forms part of the exit (E) site on the 40S ribosomal subunit and is essential for yeast viability. Human rpS5 is 67% identical and 79% similar to Saccharomyces cerevisiae rpS5 but lacks a negatively charged (pI approximately 3.27) 21 amino acid long N-terminal extension that is present in fungi. Here we report that replacement of yeast rpS5 with its human homolog yielded a viable yeast strain with a 20%-25% decrease in growth rate. This replacement also resulted in a moderate increase in the heavy polyribosomal components in the mutant strain, suggesting either translation elongation or termination defects, and in a reduction in the polyribosomal association of the elongation factors eEF3 and eEF1A. In addition, the mutant strain was characterized by moderate increases in +1 and -1 programmed frameshifting and hyperaccurate recognition of the UAA stop codon. The activities of the cricket paralysis virus (CrPV) IRES and two mammalian cellular IRESs (CAT-1 and SNAT-2) were also increased in the mutant strain. Consistently, the rpS5 replacement led to enhanced direct interaction between the CrPV IRES and the mutant yeast ribosomes. Taken together, these data indicate that rpS5 plays an important role in maintaining the accuracy of translation in eukaryotes and suggest that the negatively charged N-terminal extension of yeast rpS5 might affect the ribosomal recruitment of specific mRNAs.

Medical student research exposure via a series of modular research programs.

BACKGROUND: The falling percentage of doctors of medicine applying for National Institute of Health-funded research grants is 1 indicator that physician-scientists are a disappearing breed. This is occurring at a time when increased translational, disease-oriented, patient-oriented, and clinical research are national goals. One of the keys to providing sufficient numbers of physician-scientists to support this goal is the active targeting of medical students. We hypothesize that an improved research program infrastructure and responsiveness to changing student needs will increase student participation in research-oriented electives. METHODS: We have developed a student research program consisting of 2 Students Interested in Research noncredit electives (lecture and laboratory based), summer fellowships, support for year-out fellowships, and a Distinction in Research program that spans undergraduate medical education. Student participation and short-term research outcomes from fall 2004 through spring 2008 are analyzed to examine program efficacy. RESULTS: Students involved in the early parts of the program initially experienced higher application and success rates for summer funding opportunities, but as the program has matured, these rates have fallen in line with the class average. Independently, students participating in later portions of the program increasingly submit or publish a first author paper and have taken a year off for research during medical school. Overlap of participation in the programs is generally smaller than expected. CONCLUSION: Although structured programs can provide step-wise research experiences of increasing intensity, students may not experience a training pipeline in which each stage relies on those before and after, and instead may sample an a la carte selection of research-based enrichment opportunities.

Proteasome-mediated degradation of cotranslationally damaged proteins involves translation elongation factor 1A.

Rad23 and Rpn10 play synergistic roles in the recognition of ubiquitinated proteins by the proteasome, and loss of both proteins causes growth and proteolytic defects. However, the physiological targets of Rad23 and Rpn10 have not been well defined. We report that rad23Delta rpn10Delta is unable to grow in the presence of translation inhibitors, and this sensitivity was suppressed by translation elongation factor 1A (eEF1A). This discovery suggested that Rad23 and Rpn10 perform a role in translation quality control. Certain inhibitors increase translation errors during protein synthesis and cause the release of truncated polypeptide chains. This effect can also be mimicked by ATP depletion. We determined that eEF1A interacted with ubiquitinated proteins and the proteasome following ATP depletion. eEF1A interacted with the proteasome subunit Rpt1, and the turnover of nascent damaged proteins was deficient in rpt1. An eEF1A mutant (eEF1A(D156N)) that conferred hyperresistance to translation inhibitors was much more effective at eliminating damaged proteins and was detected in proteasomes in untreated cells. We propose that eEF1A is well suited to detect and promote degradation of damaged proteins because of its central role in translation elongation. Our findings provide a mechanistic foundation for defining how cellular proteins are degraded cotranslationally.

A birth-to-death view of mRNA from the RNA recognition motif perspective.

RNA binding proteins are a large and varied group of factors that are the driving force behind post-transcriptional gene regulation. By analogy with transcription factors, RNA binding proteins bind to various regions of the mRNAs that they regulate, usually upstream or downstream from the coding region, and modulate one of the five major processes in mRNA metabolism: splicing, polyadenylation, export, translation and decay. The most abundant RNA binding protein domain is called the RNA Recognition Motif (RRM)1. It is probably safe to say that an RRM-containing protein is making some contact with an mRNA throughout its existence. The transcriptional counterpart would likely be the histones, yet the multitude of specific functions that are results of RRM based interactions belies the universality of the motif. This complex and diverse application of a single protein motif was used as the basis to develop an advanced graduate level seminar course in RNA:protein interactions. The course, utilizing a learner-centered empowerment model, was developed to dissect each step in RNA metabolism from the perspective of an RRM containing protein. This provided a framework to discuss the development of specificity for the RRM for each required process.