Shigella flexneri is a bacterial pathogen which causes dysentery by invading the epithelial cells of the colon. In order to survive and replicate inside the host, S. flexneri requires many genes present on both its chromosome and the large virulence plasmid it carries. This study examines which genes are required for infection of cultured epithelial cells in order to understand which processes are used by S. flexneri during the infection process. This analysis pinpointed genes involved in metabolism, LPS synthesis, protein homeostasis and virulence effector proteins. The role of Elongation factor P (EF-P) in S. flexneri virulence is also investigated in this study. EF-P is a bacterial translation factor that is post-translationally modified with a [Beta]-lysine by the action of PoxA. Here it is shown that both EF-P and PoxA are necessary for virulence of S. flexneri. Loss of either EF-P or PoxA leads to an impaired ability of S. flexneri to invade epithelial cells. Proteomic analysis of efp and poxA deletion mutants revealed decreased levels of several virulence effector proteins, as well as proteins for the biosynthesis of the siderophore aerobactin. Virulence proteins were affected due to decreased levels of the master virulence regulator VirF. Reduction in VirF transcription is likely due to decreased levels of CpxA, which activates virF through the response regulator CpxR. The role of CpxAR in reduced synthesis of VirF and its downstream effectors was confirmed by showing increased invasion when a mutation resulting in constitutively vii activated CpxR was introduced into the efp mutant. Thus, modified EF-P is one of the chromosomal factor necessary for the virulence of this bacterial pathogen. / text

Diss. (sammanfattning) Stockholm : Stockholms universitet, 2008. / Härtill 4 uppsatser.

No description available.

Coding sequences (5,334 nt total) from elongation factor-1α, elongation factor-2, and the largest subunit of RNA polymerase II were determined for 6 species of Tardigrada, 2 of Arthropoda, and 2 of Onychophora. Parsimony and likelihood analyses of nucleotides and amino acids yielded strong support for Tardigrada and all internal nodes (i.e., 100% bootstrap support for Tardigrada, Eutardigrada, Parachela, Hypsibiidae, and Macrobiotidae). Results are in agreement with morphology and an earlier molecular study based on analysis of 18S ribosomal sequences. Divergence times have been estimated from amino acid sequence data using an empirical Bayesian statistical approach, which does not assume a strict molecular clock. Divergence time estimates are pre-Vendian for Tardigrada/Arthropoda, Vendian or earlier for Eutardigrada/Heterotardigrada, Silurian to Ordovician for Parachela/Apochela, Permian to Carboniferous for Hypsibiidae and Macrobiotidae, and Mesozoic for Isohypsibius/Thulinia (both within Hypsibiidae) and Macrobiotus/Richtersius (both within Macrobiotidae).

No description available.

In mammals, there are two isoforms of eukaryotic translation elongation factor 1A (eEF1A) called eEF1A1 and eEF1A2. They share 98% similarity at the amino acid level, and the main function of both is to facilitate the elongation process in protein translation. However, they have very different expression patterns. While eEF1A1 is universally expressed, eEF1A2 is strictly expressed in muscle, brain and heart. The over-expression of eEF1A2 has been found in cancers, such as ovarian and breast cancer. The factors influencing the different expression patterns of the two isoforms and the mechanisms by which eEF1A2 can act as an oncogene are not clear, therefore, the main aim of this study was to further investigate these two areas. The first aim was to find out whether the resveratrol induced down-regulation of eEF1A2 was mediated by miR-663. Western blotting in MCF7 cells showed that the level of endogenous eEF1A2 was decreased after resveratrol treatment while eEF1A1 remained stable. In contrast, NIH-3T3 stable cell lines which stably express the eEF1A2 coding sequence (CDS) only did not show this down-regulation, suggesting that the untranslated regions (UTRs) might play a role in this regulation. I then showed that miR-663 has ability to down-regulate a reporter linked to the UTRs of eEF1A2. The same reporter gene harbouring UTRs in which the binding sites of miR-663 had been deleted also showed down-regulation after resveratrol treatment, suggesting that the UTRs of EEF1A2 are key to the down-regulation of eEF1A2 by resveratrol but that miR-663 does not mediate this decrease. The second project aimed to address why eEF1A2 is an oncogene but eEF1A1 is not. The 3D structure of human eEF1A1 and eEF1A2 shows that the most of the highly conserved amino acids differences between the two isoforms are Ser and Thr residues, which are potential sites for phosphorylation. I mutated these three sites in eEF1A2 expression constructs to the equivalent amino acid from eEF1A1. Firstly, by transient transfection, all the mutant eEF1A2 were shown expressed and the sub-cellular locations of eEF1A2 remain unchanged after site-directed mutagenesis. Then, stable cell lines were generated. Anchorage independent growth (soft agar) and focus formation assays showed that the stable cell lines harbouring wild type eEF1A2 were significantly more transformed that those expressing the eEF1A2 mutants. However, there was no apparent difference in global protein synthesis between these cell lines. The results suggest that the potential phosphorylated sites in eEF1A2 play an important role in its oncogenicity and that this oncogenicity is not related to the canonical function of eEF1A2.

Eukaryotic Elongation Factor 1A (eEF1A) exists in two forms in vertebrates. The first form, eEF1A1, is expressed ubiquitously throughout development but is downregulated postdevelopmentally and replaced with eEF1A2, an isoform sharing 92% amino acid identity, in neurons and muscle. The primary function of eEF1A is to recruit amino-acylated tRNAs in a GTP-dependent manner to the A site of the ribosome during protein translation, but it also has non-canonical roles in the cell, some of which are isoform dependent. The reasons for the cell-type dependent switch from eEF1A1 to eEF1A2 are poorly understood. The first aim of this project was to examine the role played by eEF1A isoforms in neuritogenesis. To do this I used RNAi to significantly reduce expression of one or other isoform in neuronal cells and measure the effects this had on neurite outgrowth. Neurite outgrowth was significantly reduced in cells depleted of eEF1A1, but not eEF1A2. The complete loss of eEF1A2 is fatal, as has been demonstrated in the wasted mouse, an eEF1A2-null model characterised by muscle wastage, neurodegeneration and death at 4 weeks of age. Mice heterozygous for the wasted mutation have normal motor function. Recent work has found that heterozygous missense mutations in eEF1A2 can cause epilepsy and intellectual disability. It is not yet known whether the seven different de novo mutations identified to date confer loss or gain of function – a crucial piece of information required before possible treatments can be sought. The second aim of this project therefore was to investigate the role of eEF1A2 in epilepsy and intellectual disability. I achieved this by using CRISPR in two ways; firstly to model one of the mutations, D252H, in vitro in a neuronal cell line, and secondly to model another of the mutations, G70S, in vivo. No mice that recapitulated the human disease condition of EEF1A2G70S/+ were obtained however, due to the error-prone nature of the non-homologous end joining repair pathway activated by CRISPR-mediated DNA cleavage, 17 of the 35 mice born were found to be homozygous nulls at the Eef1a2 locus. Nine of these had fatal audiogenic seizures caused by sudden loud noises within the animal unit. Three mice were Eef1a2G70S/- and one Eef1a2G70S/G70S but these nonetheless showed a wasted phenotype, indicating that this mutant form of eEF1A2 has compromised function, at least in terms of translation elongation. Whether it has a toxic function ca not yet be known, but the severity of the phenotype in the G70S homozygous animal could suggest a gain of function. In in vitro experiments with exogenous eEF1A2 carrying the epilepsy-causing mutation R423C, protein expression of the mutant construct in immortalised cell lines was significantly higher when cotransfected with the wildtype construct, which mirrors the condition in humans, than when transfected alone, so the mutant protein could be stabilised in the presence of wildtype eEF1A2. I used CRISPR on LUHMES cells to make a mutant neuronal cell line containing the D252H mutation in eEF1A2. Due to time restraints no phenotypic differences between the wild type line and the D252H mutation line have yet been identified, but would form the focus of a future project.

In 1975, Elongation Factor P (EF-P) protein was first discovered in the bacterium Escherichia coli. EF-P is believed to facilitate the translation of proteins by stimulating peptide bond synthesis for a number of different aminoacyl-tRNA molecules in conjunction with the 70S ribosome peptidyl transferase. Known eukaryotic homologs, eukaryotic translation initiation factor 5A (eIF-5A) of EF-P exist but with very low sequence conservation. Nevertheless, because of the high sequence similarities seen between bacterial EF-Ps and its low sequence similarity with eIF-5A, there is interest in the pharmaceutical industry of developing a novel antibacterial drug that inhibits EF-P. Of 322 completely sequenced bacterial genomes stored in GenBank, only one organism lacked an EF-P protein. Interestingly, sixty-six genomes were discovered to carry a duplicate copy of efp. The EF-P sequences were then used to construct a protein phylogenetic tree, which provided evidence of horizontal and vertical gene transfer as well as gene duplication. To lend support to these findings, EF-P GC content, codon usage, and nucleotide and amino acid sequences were analyzed with positive and negative controls. The adjacent 10 kb upstream and downstream regions of efp were also retrieved to determine if gene order is conserved in distantly related species. While gene order was not preserved in all species, two interesting trends were seen in some of the distantly related species. The EF-P gene was conserved beside Acetyl-CoA carboxylase genes, accB and accC in certain organisms. In addition, some efp sequences were flanked by two insertion sequence elements. Evidence of gene duplication and horizontal transfers of regions were also observed in the upstream and downstream regions of efp. In combination, phylogenetic, sequence analyses, and gene order conservation confirmed evidence of the complex history of the efp genes, which showed incongruencies relative to the universal phylogenetic tree. To determine how efp is regulated, the upstream regions of efp were used to try to predict motifs in silico. While statistically significant motifs were discovered in the upstream regions of the orthologous efp genes, no conclusive similarities to known binding sites such as the sigma factor binding sites or regulatory protein binding sites were observed. This work may facilitate and enhance the understanding of the regulation, conservation, and role of EF-P in protein translation.

In 1975, Elongation Factor P (EF-P) protein was first discovered in the bacterium Escherichia coli. EF-P is believed to facilitate the translation of proteins by stimulating peptide bond synthesis for a number of different aminoacyl-tRNA molecules in conjunction with the 70S ribosome peptidyl transferase. Known eukaryotic homologs, eukaryotic translation initiation factor 5A (eIF-5A) of EF-P exist but with very low sequence conservation. Nevertheless, because of the high sequence similarities seen between bacterial EF-Ps and its low sequence similarity with eIF-5A, there is interest in the pharmaceutical industry of developing a novel antibacterial drug that inhibits EF-P. Of 322 completely sequenced bacterial genomes stored in GenBank, only one organism lacked an EF-P protein. Interestingly, sixty-six genomes were discovered to carry a duplicate copy of efp. The EF-P sequences were then used to construct a protein phylogenetic tree, which provided evidence of horizontal and vertical gene transfer as well as gene duplication. To lend support to these findings, EF-P GC content, codon usage, and nucleotide and amino acid sequences were analyzed with positive and negative controls. The adjacent 10 kb upstream and downstream regions of efp were also retrieved to determine if gene order is conserved in distantly related species. While gene order was not preserved in all species, two interesting trends were seen in some of the distantly related species. The EF-P gene was conserved beside Acetyl-CoA carboxylase genes, accB and accC in certain organisms. In addition, some efp sequences were flanked by two insertion sequence elements. Evidence of gene duplication and horizontal transfers of regions were also observed in the upstream and downstream regions of efp. In combination, phylogenetic, sequence analyses, and gene order conservation confirmed evidence of the complex history of the efp genes, which showed incongruencies relative to the universal phylogenetic tree. To determine how efp is regulated, the upstream regions of efp were used to try to predict motifs in silico. While statistically significant motifs were discovered in the upstream regions of the orthologous efp genes, no conclusive similarities to known binding sites such as the sigma factor binding sites or regulatory protein binding sites were observed. This work may facilitate and enhance the understanding of the regulation, conservation, and role of EF-P in protein translation.

The GTPase elongation factor G (EF-G) is an important player in the complex process of protein synthesis by bacterial ribosomes. Although extensively studied much remains to be learned about this fascinating protein. In the elongation phase, after incorporation of each amino acid into the growing peptide chain, EF-G translocates the ribosome along the mRNA template. In the recycling phase, when the synthesis of a protein has been completed, EF-G, together with ribosome recycling factor (RRF), splits the ribosome into its subunits. We developed the first in vitro assay for measuring the average time of a complete translocation step at any position along the mRNA. Inside the open reading frame, at saturating EF-G concentration and low magnesium ion concentration, translocation rates were fast and compatible with elongation rates observed in vivo. We also determined the complete kinetic mechanism for EF-G- and RRF-dependent splitting of the post-termination ribosome. We showed that splitting occurs only when RRF binds before EF-G and that the rate and GTP consumption of the reaction varies greatly with the factor concentrations. The antibiotic fusidic acid (FA) inhibits bacterial protein synthesis by binding to EF-G when the factor is ribosome bound, during translocation and ribosome recycling. We developed experimental methods and a theoretical framework for analyzing the effect of tight-binding inhibitors like FA on protein synthesis. We found that FA targets three different states during each elongation cycle and that it binds to EF-G on the post-termination ribosome both in the presence and absence of RRF. The stalling time of an FA-inhibited ribosome is about hundred-fold longer than the time of an uninhibited elongation cycle and therefore each binding event has a large impact on the protein synthesis rate and may induce queuing of ribosomes on the mRNA. Although ribosomes in the elongation and the recycling phases are targeted with similar efficiency, we showed that the main effect of FA in vivo is on elongation. Our results may serve as a basis for modelling of EF-G function and FA inhibition inside the living cell and for structure determination of mechanistically important intermediate states in translocation and ribosome recycling.