Yeast Elongator was initially isolated based on its interaction with the hyper-phosphorylated form of RNA polymerase II. Later on, it was shown to possesion intrinsic histone acetyl-transferase activity and to crosslink to nascent RNA in vivo. The six ELP genes were also identified, among other genes, in a genetic screen in Saccharomyces cerevisiae for targets of the toxin zymocin. KTI12, which stands for *jC lactis toxin Insensitive 12", was one of those other genes. ktil2A mutant cells display phenotypes closely resembling those of elpA mutants. Moreover, Ktil2 protein was shown to co-immunoprecipitate with Elongator and with RNA polymerase II, indicating a functional interaction with both factors. The experiments presented in the first part of this thesis confirm genetically and biochemically that Elongator is a six-subunit complex. In vitro and in vivo studies were performed to reveal the molecular architecture of this complex. Briefly, strong pair-wise interactions between Elpl and Elp3, Elp4 and Elp6 and between Elp5 and Elp6 were uncovered. Additionally, a weak interaction between Elp3 and Elp4 was observed. In vitro HAT assays and RNA immunoprecipitation experiments suggested that Elp2 is dispensable for the in vitro histone acetyl-transferase and the in vivo RNA binding activity of Elongator. In contrast, Elp3 was shown to be critical for both the integrity of the complex and its in vivo RNA binding activity. Moreover, the localisation of yeast Elp3 protein was studied, in an attempt to address the possibility that Elongator continuously shuttles from the cytoplasm to the nucleus. In the second part, biochemical and genetic studies strongly suggested that Ktil2 interacts with Elongator and might regulate its in vivo HAT activity. Finally, the molecular mechanism of action of the toxin zymocin was studied. In particular, preliminary experiments, which test the possibility that the RNAPII gets degraded in response to zymocin treatment of 5. cerevisiae cells, are presented.

No description available.

No description available.

Toxicity of the zymocin complex from Kluyveromyces lactis, which causes cell death of Saccharomyces cerevisiae, relies on a specific tRNA anticodon modification in sensitive yeast cells. Primarily tRNAGlu with a highly modified uridine base in the anticodon wobble position (U34) serves as recognition and cleavage site for zymocin, which functions as a tRNA endoribonuclease. Cleavage results in depletion of essential tRNAs, translational breakdown and ultimately cell death. KTI11, URM1 and SIT4 are genes which confer zymocin sensitivity. Interestingly, deletion of each of these genes protects against zymocin due to U34 modification defects. Here, using genetic analysis, data was generated to further our understanding about how KTI11, URM1 and SIT4 function in the process of tRNA modification and zymocin dependent tRNA-cleavage. This work shows that Kti11 associates with two different protein complexes involved in translationally relevant modification pathways. Kti11 partners with the multifunctional Elongator complex and this association is crucial for Elongator functions in tRNA modification, explaining why kti11 mutants phenocopy Elongator minus cells. Furthermore, evidence is provide that Kti11 is subunit of a trimeric complex, Dph1•Dph2•Kti11, which is required to form a unique diphthamide modification on eEF2. Moreover, based on the zymocin resistance of urm1Δ cells and since Urm1 harbours features of prokaryotic sulphur carriers, it was hypothesised that Urm1 may be involved in U34 thio-modification. Data are presented suggesting that Urm1-Uba4 function as a sulphur relay system responsible for thiolation of tRNAs. Finally, this study focussed on the investigation of protein phosphatase Sit4. It is shown that Sit4 and Rrd1 form a functional phosphatase complex supporting Sit4 functions in the Target Of Rapamycin (TOR) pathway. Finally, the data suggest that SIT4, URM1 and KTI11 dependent tRNA anticodon modification has a signalling function and is implicated in the Nitrogen Catabolite Repression (NCR) branch of the TOR pathway.

Aneuploidy is defined as a state of having an abnormal number of chromosomes within a cell. In human, congenital aneuploidy is the leading cause of developmental abnormality and mental retardation. Somatically acquired aneuploidy has long been known as a hallmark of cancer genome. While multiple studies have focused on dissecting the molecular mechanisms leading to aneuploidy, little has been done to address its consequences. Using budding yeast Saccharomyces cerevisiae as model organism, we first asked whether and how aneuploidy could bring about phenotypic variation. We monitored the growth of a panel of 38 isogenic and relative stable aneuploid strains under various conditions including a panel of chemotherapeutic and antifungal drugs. We found that some aneuploid strains grew significantly faster than euploid control strains in various conditions. Using quantitative proteomic, we showed that aneuploidy could induce large change to the cellular proteome and the levels of protein expression largely scale with chromosome copy numbers. Together, these findings suggest that aneuploidy, through its effect on proteome, could underlie phenotypic variation.

Global inhibition of protein synthesis is a common response to diverse stress conditions. This allows the protein synthesis machinery to be redirected towards the expression of genes required for protection against the stress. All organisms are exposed to reactive oxygen species (ROS) as by-productsarive metabolism or through exposure to radical-generatmg compounds. An oxidative stress occurs when ROS overwhelm the antioxidant defences of the cell. Here, the regulation of protein synthesis in response to oxidative stress in Saccharomyces cerevisiae was exarnmed after exposure to hydroperoxides.

The aim of this investigation was to assess the impact of yeast strain, initial glucose concentration and fermentation temperature on fermentation kinetic parameters. In this study two industrial Saccharomyces cereviaise strains were investigated, LAL 7 and Ethanol Red. The strains were hexose utilization competent but could not utilize pentoses. Tolerance was assessed in aerobic and anaerobic conditions to the following fermentation stresses: osmotic stress induced by sorbitol, NaCI and high glucose fermentations; ethanol stress; and thermal stress. It was observed that Ethanol Red was more tolerant than LAL 7 to all conditions applied. However, LAL 7 possessed a greater capacity to utilize glucose and complete attenuation of fermentation at all scales assessed. During fermentation it was noted that viability for LAL 7 was particularly impaired suggesting its suitability for pitch and ditch fermentation practices only. In contrast Ethanol Red was more robust and could potentially be applied to recycled or continuous fermentation practices. Finally a companson was made of the fermentation kinetic parameters exhibited by Ethanol Red and LAL 7 during fermentation at the 5L scale. Although the initial and final viabilities as well as growth rates of both strains differed, completion of fermentation occurred at a similar time and ethanol yields were also similar. Differences in the concentration of trehalose accumulated during fermentation were noted with Ethanol.Red producing more of this intracellular carbohydrate than LAL 7. It is proposed that this reflects the superior stress tolerance of the former and it is suggested that trehalose accumulation may be an effective stress selection criteria for strains for bioethanol fermentation.

Precise, complete and timely replication of eukaryotic genomes is a prerequisite to cell division. Each chromosome replicates in a defined temporal order that is dictated by the variable activation timings and efficiencies of replication origins. However, so far the mechanisms regulating origin activity have remained elusive. Replication origins are best understood in the budding yeast Saccharomyces cerevisiae. Powerful comparative genomic approaches are possible in budding yeasts due to the evolutionary range of sequenced genomes available and their tractability to genetic approaches. Previously, functional sequence elements at replication origins have been identified based upon their phylogenetic sequence conservation amongst closely related species of the sensu stricto group. To gain insight into the selective pressures contributing to this phylogenetic conservation, mutant strains with chromosomally inactivated origins were grown in competition with wild-type strains. Origin mutant strains did not have a growth defect compared to the wild-type, suggesting that the selective advantage conferred by evolutionary conserved origins is not the requirement for a rapid cell cycle time. To improve the reference sequence annotation, the S. cerevisiae genome was systematically screened for origin function, confirming more than 200 additional replication origins. The resulting comprehensive map of origin locations in S. cerevisiae was used to assess the accuracy of origin predictions from published studies and two newly developed techniques. These two approaches use high-throughput sequencing to either identify replication origin locations or measure replication dynamics genome-wide. Using the latter method on haploid and diploid S. cerevisiae strains showed that replication dynamics are independent of cell ploidy. Genome replication in divergent budding yeasts was investigated using a combination of replication timing profiles acquired with deep sequencing and plasmid-based assays. These analyses formed the basis for a comparative genomics approach, which revealed that the relative order of genome replication is conserved. A minority of replication origins with identical genomic locations show differences in activity between the analyzed species. To gain insight into the mechanisms underlying origin regulation, the replication dynamics of a hybrid between S. cerevisiae and its most distant relative in the sensu stricto group - S. bayanus - were measured. Replication origin function was found to be controlled by both local and global regulators .

Maintaining the correct balance of the various proteins found within a cell is a major process. Many factors come together during gene expression to transcribe a gene into mRNA and translate this transcript into a polypeptide chain. These processes are themselves formed of many stages, each of which provides points for a cell to ensure accuracy and adjust the amount of a certain protein. At the end of translation, the eukaryotic release factors eRF1 and eRF3 interact to release the nascent polypeptide chain from the ribosome. The main question remaining to be answered is how release factor levels, and with them termination accuracy, are regulated in Saccharomyces cerevisiae. This study focused on transcription and translational, as well as post translational events, as possible points of regulation. Our findings suggest that there is no feedback mechanism at either transcription or translation level which allows a cell to sense and adjust cellular levels of either of the . release factors. Although both eRF1 and eRF3 were found to be highly stable under non- stress conditions, microscopy studies highlighted the possible involvement of the yeast vacuole in regulating translation factor abundance during environmental stress. While no significant level changes were observed for the class one release factor eRF1 and several elongation factors, eRF3 levels were found to increase in protease deletion strains. Levels of the release factor were furthermore affected by deletions of the N-terminus in part or as a whole, though no connection was found linking the N-terminal region of eRF3 and the vacuole to one another in regulating cellular levels of the release factor. Moreover, this study highlighted that release factor levels, their ratio to one another and other factors involved in translation, are inextricably linked to termination accuracy. Data produced by this investigation suggests that eRF3 levels and activity are affected by a network of other factors. In specific, data presented here support studies published by other Laboratories reporting a functional interaction between eRF3 and the nucleotide exchange factor eEF1B and suggest that eRF1 might act as TDI for eRF3, as seen in mammalian cells.

Prions are classically understood as infectious agents capable of transmission by a mechanism of conformational templating where native protein is converted to the prion form and as such, unlike any other transmissible agent, does not involve nucleic acid. Prions however also influence a range of cellular phenotypes in yeast and beyond without any resulting detriment to the host so can act as functional epigenetic determinants of phenotype and perhaps even drivers of evolution. The prion form of the eukaryotic release factor Sup35p forms the [Pst] prion which amongst other effects, leads to adoption of a nonsense suppression phenotype. The as yet poorly understood entity responsible for maintaining the [Pst] phenotype in a dividing population has been termed the propagon. Although [Pst] is attributable to prionised Sup35p material, which assembles into high molecular weight amyloid like structures, no specific structural species has been specifically credited with propagon activity. The following work describes optimised methods for use in characterising key properties of the [Pst] system relating to propagation, and applies them along with novel new approaches to investigate the molecular nature of the propagon. Prion systems may be capable of adapting cell populations to changes in environment however how this is achieved is unclear. Propagon function as a modulator of phenotype was investigated in response to glucose exhaustion, key changes to the prion system were observed and characterised and the ro le of prions systems in cell ular responses to environment discussed. Size appears to be a defining property of aggregate transmissibility however how size influences propagon activity beyond affecting diffusion properties is unclear. Organisation of low molecular weight species into high molecular weight aggregates provides a clear determinant of size however very little is understood about the nature or significance of such organisation. The fo llowing work explores the molecular nature and biological relevance of these two species and discusses how they may influence our understanding of the [PSI] system.