Protein eIF4E2 belongs to the family of eukaryotic translation initiation factors 4E, but it does not participate in translation initiation under normal circumstances. Its main role lies in translational repression of specific mRNAs. Nevertheless eIF4E2 takes part in translation initiation as a subunit of a specific translation initiation complex in hypoxic conditions. The exact mechanism in which eIF4E2 takes part in either of these processes is not known. One way to study the role of eIF4E2 in the cell is to find out what other proteins does eIF4E2 interact with. The goal of this work was to seek out potential eIF4E2-interacting partners in the HEK293 cell line using immunoprecipitation followed by mass spek- trometry. Apart from finding individual proteins the goal was to identify eIF4E2-containig protein com- plexes in HEK293 cells. A second line of work was preparation of a system for screening inhibitors of the interaction between eIF4E2 and eIF4G3. The main result is finding potential new eIF4E2-intera- cting partners in human cells.

The process of translation initiation is the most highly regulated step of protein synthesis. In bacteria, three initiation factors (IF1, IF2 and IF3) play crucial roles during initiation. IF3 acts as an anti-association factor for the two ribosomal subunits. Eubacterial IF3 also permits initiator tRNA (i-tRNA) selection at the P site of the ribosome. Two features of i-tRNA, i. e. the characteristic 3GC base pairs in the anticodon stem and the cognate interaction of the anticodon sequence with the initiation codon of the mRNA contribute to IF3 based selection and/or proofreading. However, the exact mechanism of this discrimination and the contribution of the individual domains towards this process of selection/ proofreading are unclear. Further, there are exceptional instances in the natural world where either the codon-anticodon interaction or the anticodon stem composition deviates from the norm. For instance, in mammalian mitochondria, non-AUG codons such as AUU and AUA are present in the genome although they are notoriously poor initiation codons. In addition, some species of Mycoplasma have i-tRNAs with variations in the typically conserved 3GC base pairs of the anticodon stem. In this study, we have investigated the mechanism of proofreading activity of IF3 of E. coli, mitochondrial and mycoplasmal origins. Part I: Proofreading function of IF3 in E. coli IF3 is composed of N and C terminal domains joined by a flexible linker region. By means of complete and partial IF3 knockouts, we show that the C-terminal domain (CTD) is essential for the survival of E. coli while the N-terminal (NTD) is required for cellular fitness. Using reporter assays, we have established the role of the NTD in proofreading, while polysome profile analyses reaffirm that the CTD alone can bind to the 30S and carry out ribosome anti-association. Therefore, we show that the CTD is the ribosome binding and anti-association domain, while the NTD is the major proofreading domain. Unpublished cryoEM structures from Prof. Ramakrishnan’s lab indicate that the NTD of IF3 pushes the i-tRNA at its elbow and helps in P site accommodation of the i-tRNA. We propose that when the codon-anticodon interaction is non-cognate or if the 3GC base pairs of the anticodon stem are not intact, then the dynamic action of the NTD destabilises the tRNA at the P site and leads to its rejection. Part II: Proofreading function of mitochondrial IF3 (IF3mt) Of the 13 protein-coding genes in mammalian mitochondria, 3 utilise the non-canonical AUA codon and one utilises the non-canonical start codon AUU. Since IF3mt does not possess many of the generally conserved residues implicated in proofreading, we decided to characterise the proofreading function of IF3mt and its role in initiation with non-canonical start codons. Structurally, IF3mt is similar to EcoIF3 with its N and C terminal domains joined by a linker region. However, IF3mt additionally possesses N- and C-terminal extensions which are generally disordered in structure. In vivo studies of mitochondrial translation factors have been mired by the lack of methodologies to manipulate mitochondria. We have developed an E. coli strain to study the proofreading functions of mitochondrial IF3 (IF3mt) with the help of reporter genes. Consistent with its function in mitochondria, IF3mt allowed promiscuous initiation from non-AUG codons. However, IF3mt avoided initiation with i-tRNAs lacking evolutionarily conserved 3GC pairs in anticodon stems. Interestingly, expression of IF3mt N-terminal domain or IF3mt devoid of its typical N-, and C-terminal extensions significantly improved its proofreading activity. Our immunoblot assays from polysome profile fractions indicate that the IF3mt derivative lacking extensions is capable of superior 30S ribosome binding. The two derivatives of IF3mt missing the Next (IF3mtΔNext) or both the Next and Cext (IF3mtΔNextCext) display an affinity for the 50S ribosome. We propose that the extensions of IF3mt may have evolved to reduce the affinity of IF3mt to the ribosome and thereby permit initiation with non-canonical start codons like AUU and AUA. Our studies suggest that E. coli provides an excellent heterologous model to study distinctive features of mitochondrial factors. Part III: Fidelity of translation initiation in mycoplasma One of the many singular features of mycoplasma is the presence of many anticodon stem variants of the i-tRNA across different species. In general, i-tRNAs are characterized by the presence of the typical feature of the conserved 3 consecutive GC base pairs (GC/GC/GC) in the anticodon stem. However, many mycoplasmal species have i-tRNAs with AU/GC/GC, GC/GC/GU or AU/GC/GU sequences. Interestingly, the mycoplasmal species which harbour the AU/GC/GU i-tRNA are also human pathogens. Therefore, we decided to investigate whether these organisms possess any unique features to accommodate the i-tRNA variants, by investigating the usage of Shine Dalgarno sequences and by carrying out multiple sequence alignments of genes encoding initiation factors, ribosomal proteins S9 and S13 and 16S rRNA. Since IF3 plays a crucial role in i-tRNA selection, we carried out computational analysis of mycoplasmal IF3 sequences, which revealed many interesting features. Most striking amongst them was the variation of the highly conserved R at position 131 in some species. Interestingly, these were the very mycoplasmal species which possessed the anticodon stem variant AU/GC/GU, suggesting a strong correlation between these two features. It is known that the R131P mutation of EcoIF3 is characterised by an enormous loss of proofreading activity. It seemed unusual that such compromised proofreading would be tolerated in the cell, so we decided to investigate other components of the translational machinery as well. The C-terminal SKR tail of the ribosomal protein S9, which contacts the P-site tRNA, is highly conserved across bacteria. Analysis of the C-terminal sequences of S9 proteins in various mycoplasmal species revealed a surprising variation- the presence of a TKR tail in strains with the AU/GC/GU tRNA. In this study we have investigated the co-occurrence of S9 and IF3 variations in i-tRNA selection in E. coli. We see that the R131P polymorphism of IF3 leads to a tremendous loss of proofreading, but this loss is significantly tempered by the presence of the S9 TKR variation. Our bioinformatics studies revealed that the mycoplasmal species which are sustained on AU/GC/GU i-tRNAs also tend to use a higher percentage of non-AUG codons. By means of our reporter assays in E. coli, we have shown once again that the R131P polymorphism of IF3 leads to a tremendous increase in initiation with the non-canonical start codon AUA, but this increase is significantly tempered by the presence of the S9 TKR variation.

The ability to sense and respond to environmental cues is crucial for the survival of all organisms. This response is often manifested by exerting control at different levels of gene expression, i.e. transcription, translation and post translation levels. Global control of protein synthesis is frequently exercised at the initial step of translation initiation and is generally achieved by changes in the phosphorylation state of initiation factors or the regulators that interact with them. The formation of ternary complex (TC) is considered first step of translation initiation and depends on the recycling of inactive eIF2-GDP to active eIF2-GTP form. This nucleotide exchange reaction is catalyzed by the eukaryotic initiation factor-2B (eIF2B). eIF2B is composed of a regulatory sub-complex of alphaβdelta subunits and a catalytic sub-complex of the γε subunits. The guanine nucleotide exchange activity of eIF2B is regulated by phosphorylation of eIF2alpha and additionally in mammalian cells, by direct phosphorylation of eIF2B at multiple sites in ε subunit, where most of the catalytic activity of eIF2B resides. Recent unpublished studies in the Pavitt laboratory identified novel phosphorylation sites by Mass Spectrometry in γ and ε subunits of eIF2B catalytic sub-complex. In order to study the functional significance of these phospho-sites for translation initiation, Site Directed Mutagenesis (SDM) was performed to generate Ser to Ala mutants. All mutations are viable and have no significant growth defect on rich or minimal media; however the significance of these sites in yeast growth became apparent by growing yeast in different stress conditions (e.g. Rapamycin, Torin1, amino acid starvation and 1-butanol). Effects on the phosphorylation pattern at these sites were monitored by using custom generated phospho-specific antibodies. All phosphorylation events appear independent of the eIF2alpha kinase (Gcn2p in yeast). The phosphorylation of ε-S528 depends on the presence of ε-S525. This study finds that addition of rapamycin, Torin1, amino acid starvation and butanol, which each inhibits global translation initiation, alters the phosphorylation pattern at ε-S435, ε-S525 and ε-S528 sites. Linking growth to phosphorylation, it appears that phosphorylation at ε-S435 and ε-S525 is directly proportional to growth. Phosphorylation of ε-S435 is necessary for effect of eIF2alpha-Ser51 phosphorylation on protein synthesis while phosphorylation of ε-S528 seems to be a target of various mechanisms. This study also suggests that eIF2Bε may be a key player of the cell cycle progression and phosphorylation changes can serve as marker for the regulation of eIF2B activity. The kinases responsible for phosphorylation at these sites are not yet known in yeast. Further investigation is required to find the functional significance of alterations in phosphorylation pattern to definitively establish eIF2Bε phosphorylation as a mechanism for regulating eIF2B activity in yeast. Models are presented to account for the results obtained that show how phosphorylation of eIF2Bε at these sites may contribute to the control of protein synthesis.

The S1 genome segment of avian reovirus is functionally tricistronic, encoding three independent protein products (named p10, p17 and ?C) from three sequential, partially overlapping open reading frames (ORFs). The dogma of translation initiation, the cap-dependent scanning model, suggests that ribosomes would normally only translate the 5?-proximal ORF. Four alternate mechanisms of translation initiation could account for translation of the downstream ?C ORF; an IRES element, reinitiation, ribosome shunting, and leaky scanning. The objective of my doctoral research was to investigate the translation initiation mechanisms that are operative on the S1 mRNA. Translation of the p10 and p17 ORFs was revealed to be coordinated via standard leaky scanning, while none of the known mechanisms of translation initiation could account for expression of the ?C ORF. Further investigation determined that two alternate cap-dependent mechanisms contribute to translation initiation at the ?C AUG codon. The first mechanism involves a modified version of enhanced leaky scanning. Although insertion of upstream elements known to impede scanning ribosomal subunits dramatically inhibited translation of the downstream ORF in the context of other mRNAs, the same elements only marginally reduced ?C translation. Specific features of the S1 mRNA therefore function to promote leaky scanning and translation of the ?C ORF. The inability to eliminate ?C expression beyond a threshold retention level of ~20-30%, despite the presence of eight upstream start codons that should eliminate leaky scanning, strongly suggests that ribosomes must also utilize a scanning-independent means to access the internal ?C start site. This mechanism for ?C translation initiation, which I termed ribosome handoff, allows ribosomes to bypass upstream elements, and requires a sequence-dependent translation enhancer element present within S1 nucleotides 366-392 that may function to mediate handoff via complementarity with 18S ribosomal RNA. Translation initiation at the ?C start site is therefore made possible by two alternative mechanisms, enhanced leaky scanning and ribosome handoff from the 5?-cap. The novelty of these two mechanisms highlights the complexity of the translation initiation process and the potential heterogeneity of cellular ribosomes, which raises the possibility that internal initiation may be far more common than currently appreciated.

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