Comparison of the ability of mammalian eEF1A1 and its oncogenic variant eEF1A2 to interact with actin and calmodulin

The question as to why a protein exerts oncogenic properties is answered mainly by well-established ideas that these proteins interfere with cellular signaling pathways. However, the knowledge about structural and functional peculiarities of the oncoproteins causing these effects is far from comprehensive. The 97.5% homologous tissue-specific A1 and A2 isoforms of mammalian translation elongation factor eEF1A represent an interesting model to study a difference between protein variants of a family that differ in oncogenic potential. We propose that the different oncogenic impact of A1 and A2 might be explained by differences in their ability to communicate with their respective cellular partners. Here we probed this hypothesis by studying the interaction of eEF1A with two known partners – calmodulin and actin. Indeed, an inability of the A2 isoform to interact with calmodulin is shown, while calmodulin is capable of binding A1 and interferes with its tRNA-binding and actin-bundling activities in vitro. Both A1 and A2 variants revealed actin-bundling activity; however, the form of bundles formed in the presence of A1 or A2 was distinctly different. Thus, a potential inability of A2 to be controlled by Ca2+-mediated regulatory systems is revealed

A tryptophan-rich peptide acts as a transcription activation domain

<p>Abstract</p> <p>Background</p> <p>Eukaryotic transcription activators normally consist of a sequence-specific DNA-binding domain (DBD) and a transcription activation domain (AD). While many sequence patterns and motifs have been defined for DBDs, ADs do not share easily recognizable motifs or structures.</p> <p>Results</p> <p>We report herein that the N-terminal domain of yeast valyl-tRNA synthetase can function as an AD when fused to a DNA-binding protein, LexA, and turn on reporter genes with distinct LexA-responsive promoters. The transcriptional activity was mainly attributed to a five-residue peptide, WYDWW, near the C-terminus of the N domain. Remarkably, the pentapeptide <it>per se </it>retained much of the transcriptional activity. Mutations which substituted tryptophan residues for both of the non-tryptophan residues in the pentapeptide (resulting in W₅) significantly enhanced its activity (~1.8-fold), while mutations which substituted aromatic residues with alanine residues severely impaired its activity. Accordingly, a much more active peptide, pentatryptophan (W₇), was produced, which elicited ~3-fold higher activity than that of the native pentapeptide and the N domain. Further study indicated that W₇mediates transcription activation through interacting with the general transcription factor, TFIIB.</p> <p>Conclusions</p> <p>Since W₇shares no sequence homology or features with any known transcription activators, it may represent a novel class of AD.</p

Modeling Translation in Protein Synthesis with TASEP: A Tutorial and Recent Developments

The phenomenon of protein synthesis has been modeled in terms of totally asymmetric simple exclusion processes (TASEP) since 1968. In this article, we provide a tutorial of the biological and mathematical aspects of this approach. We also summarize several new results, concerned with limited resources in the cell and simple estimates for the current (protein production rate) of a TASEP with inhomogeneous hopping rates, reflecting the characteristics of real genes.Comment: 25 pages, 7 figure

Synonymous Codon Ordering: A Subtle but Prevalent Strategy of Bacteria to Improve Translational Efficiency

Background: In yeast coding sequences, once a particular codon has been used, subsequent occurrence of the same amino acid tends to use codons sharing the same tRNA. Such a phenomenon of co-tRNA codons pairing bias (CTCPB) is also found in some other eukaryotes but it is not known whether it occurs in prokaryotes. Methodology/Principal Findings: In this study, we focused on a total of 773 bacterial genomes to investigate their synonymous codon pairing preferences. After calculating the actual frequencies of synonymous codon pairs and comparing them with their expected values, we detected an obvious pairing bias towards identical codon pairs. This seems consistent with the previously reported CTCPB phenomenon, since identical codons are certainly read by the same tRNA. However, among co-tRNA but non-identical codon pairs, only 22 were often found overrepresented, suggesting that many co-tRNA codons actually do not preferentially pair together in prokaryotes. Therefore, the previously reported co-tRNA codons pairing rule needs to be more rigorously defined. The affinity differences between a tRNA anticodon and its readable codons should be taken into account. Moreover, both within-gene-shuffling tests and phylogenetic analyses support the idea that translational selection played an important role in shaping the observed synonymous codon pairing pattern in prokaryotes. Conclusions: Overall, a high level of synonymous codon pairing bias was detected in 73 % investigated bacterial species

Phenotypic Screen of Early-Developing Larvae of the Blood Fluke, Schistosoma mansoni, using RNA Interference

RNA interference (RNAi) represents the only method currently available for manipulating gene-specific expression in Schistosoma spp., although application of this technology as a functional genomic profiling tool has yet to be explored. In the present study 32 genes, including antioxidants, transcription factors, cell signaling molecules and metabolic enzymes, were selected to determine if gene knockdown by RNAi was associated with morphologically definable phenotypic changes in early intramolluscan larval development. Transcript selection was based on their high expression in in vitro cultured S. mansoni primary sporocysts and/or their potential involvement in developmental processes. Miracidia were allowed to transform to sporocysts in the presence of synthesized double-stranded RNAs (dsRNAs) and cultivated for 7 days, during which time developing larvae were closely observed for phenotypic changes including failure/delay in transformation, loss of motility, altered growth and death. Of the phenotypes evaluated, only one was consistently detected; namely a reduction in sporocyst size based on length measurements. The size-reducing phenotype was observed in 11 of the 33 (33%) dsRNA treatment groups, and of these 11 phenotype-associated genes (superoxide dismutase, Smad1, RHO2, Smad2, Cav2A, ring box, GST26, calcineurin B, Smad4, lactate dehydrogenase and EF1α), only 6 demonstrated a significant and consistent knockdown of specific transcript expression. Unexpectedly one phenotype-linked gene, superoxide dismutase (SOD), was highly induced (∼1600-fold) upon dsRNA exposure. Variation in dsRNA-mediated silencing effects also was evident in the group of sporocysts that lacked any definable phenotype. Out of 22 nonphenotype-expressing dsRNA treatments (myosin, PKCB, HEXBP, calcium channel, Sma2, RHO1, PKC receptor, DHHC, PepcK, calreticulin, calpain, Smeg, 14.3.3, K5, SPO1, SmZF1, fibrillarin, GST28, GPx, TPx1, TPx2 and TPx2/TPx1), 12 were assessed for the transcript levels. Of those, 6 genes exhibited consistent reductions in steady-state transcript levels, while expression level for the rest remained unchanged. Results demonstrate that the efficacy of dsRNA-treatment in producing consistent phenotypic changes and/or altered gene expression levels in S. mansoni sporocysts is highly dependent on the selected gene (or the specific dsRNA sequence used) and the timing of evaluation after treatment. Although RNAi holds great promise as a functional genomics tool for larval schistosomes, our finding of potential off-target or nonspecific effects of some dsRNA treatments and variable efficiencies in specific gene knockdown indicate a critical need for gene-specific testing and optimization as an essential part of experimental design, execution and data interpretation

Distribution and Phylogeny of EFL and EF-1α in Euglenozoa Suggest Ancestral Co-Occurrence Followed by Differential Loss

BACKGROUND: The eukaryotic elongation factor EF-1alpha (also known as EF1A) catalyzes aminoacyl-tRNA binding by the ribosome during translation. Homologs of this essential protein occur in all domains of life, and it was previously thought to be ubiquitous in eukaryotes. Recently, however, a number of eukaryotes were found to lack EF-1alpha and instead encode a related protein called EFL (for EF-Like). EFL-encoding organisms are scattered widely across the tree of eukaryotes, and all have close relatives that encode EF-1alpha. This intriguingly complex distribution has been attributed to multiple lateral transfers because EFL's near mutual exclusivity with EF-1alpha makes an extended period of co-occurrence seem unlikely. However, differential loss may play a role in EFL evolution, and this possibility has been less widely discussed. METHODOLOGY/PRINCIPAL FINDINGS: We have undertaken an EST- and PCR-based survey to determine the distribution of these two proteins in a previously under-sampled group, the Euglenozoa. EF-1alpha was found to be widespread and monophyletic, suggesting it is ancestral in this group. EFL was found in some species belonging to each of the three euglenozoan lineages, diplonemids, kinetoplastids, and euglenids. CONCLUSIONS/SIGNIFICANCE: Interestingly, the kinetoplastid EFL sequences are specifically related despite the fact that the lineages in which they are found are not sisters to one another, suggesting that EFL and EF-1alpha co-occurred in an early ancestor of kinetoplastids. This represents the strongest phylogenetic evidence to date that differential loss has contributed to the complex distribution of EFL and EF-1alpha

Highly homologous eEF1A1 and eEF1A2 exhibit differential post-translational modification with significant enrichment around localised sites of sequence variation

Translation elongation factors eEF1A1 and eEF1A2 are 92% identical but exhibit non-overlapping expression patterns. While the two proteins are predicted to have similar tertiary structures, it is notable that the minor variations between their sequences are highly localised within their modelled structures. We used recently available high-throughput “omics” data to assess the spatial location of post-translational modifications and discovered that they are highly enriched on those surface regions of the protein that correspond to the clusters of sequence variation. This observation suggests how these two isoforms could be differentially regulated allowing them to perform distinct functions. REVIEWERS: This article was reviewed by Frank Eisenhaber and Ramanathan Sowdhamini

Channeling of aminoacyl-tRNA for protein synthesis in vivo.

Channeling, the direct transfer of metabolic intermediates from one enzyme to another in a pathway, has received increased attention as an explanation for the high efficiency of cellular processes. The known structural organization of the protein biosynthetic machinery, and a recent suggestion that aminoacyl-tRNAs may be channeled, has led us to devise a direct test of this possibility. By employing the technique of electroporation, conditions were established for the introduction of aminoacyl-tRNAs into Chinese hamster ovary (CHO) cells. We show, by coelectroporation of various combinations of free [14C]amino acids and [3H]aminoacyl-tRNAs, that whereas the free amino acids serve as effective precursors for protein synthesis, the exogenous aminoacyl-tRNAs are utilized poorly, if at all. The lack of incorporation into protein from added aminoacyl-tRNAs is not due to their leakage from the cell, to their instability, or to their damage during electroporation. Furthermore, in contrast to the findings with intact cells, extracts of CHO cells incorporate both free amino acids and aminoacyl-tRNAs into protein with similar efficiencies. Based on these observations, we conclude that the inability of exogenous aminoacyl-tRNAs to serve as precursors for protein synthesis is due to the structural organization of intact cells that leads to channeling of this substrate in vivo. Thus, we propose that endogenously synthesized aminoacyl-tRNA is directly transferred from aminoacyl-tRNA synthetase to elongation factor to ribosome without dissociation into the cell fluid, and as a consequence, usage of exogenously introduced molecules is precluded

A sequestered pool of aminoacyl-tRNA in mammalian cells.

We have recently proposed that aminoacyl-tRNA is channeled during protein synthesis in vivo--i.e., it is directly transferred among the components of the protein-synthesizing machinery and does not mix with aminoacyl-tRNA molecules introduced from outside the cell. To understand the structural basis for these functional properties, we have examined the disposition of aminoacyl-tRNA within the cell. To do this we have developed a Chinese hamster ovary (CHO) permeabilized-cell system using the plant glycoside saponin. We show, using a mixture of free 14C-labeled amino acids and 3H-labeled aminoacyl-tRNAs, that 14C-labeled aminoacyl-tRNAs synthesized endogenously from the free amino acids are preferentially sequestered within the cell, whereas their exogenous 3H counterparts distribute between the inside and outside of the cell based solely on the relative volumes of the two compartments. Furthermore, the endogenous 14C-labeled aminoacyl-tRNA population is resistant to pancreatic ribonuclease action, whereas the 3H molecules are rapidly degraded. We conclude, based on these observations, that aminoacyl-tRNAs synthesized in vivo are continually associated with components of the protein synthesis machinery and are thereby retained within the permeabilized cell and are also protected from RNase action. These data provide independent evidence for the channeling model of protein biosynthesis