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    Identification of large ribosomal proteins required for the full activation of the protein kinase Gcn2 in Saccharomyces cerevisiae : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biological Sciences at Massey University, Albany, New Zealand. EMBARGOED indefinitely.
    (Massey University, 2019) Anderson, Reuben Andrew
    Protein synthesis is a fundamental biological process that all organisms require for maintaining life, growth and development. The maintenance of amino acid levels, the building blocks of proteins, is essential for maintaining protein synthesis under all biological conditions. Hence, amino acid shortage can be deleterious to the cell. Therefore, cells harbour mechanisms to cope and overcome amino acid starvation. When eukaryotes are subjected to amino acid starvation, the resulting accumulation of uncharged tRNAs activates the protein kinase Gcn2, leading to phosphorylation of eIF2α and activation of the amino acid starvation response. Uncharged tRNAs are the signal of starvation, directly detected by Gcn2. Gcn2 must bind to the effector protein Gcn1 and both must contact ribosomes for Gcn2 activation. The current working model for how the starvation signal is delivered to Gcn2 postulates that these uncharged tRNAs bind in the A-site of the ribosome in a codon specific manner, which are subsequently transferred to Gcn2. Gcn1 is directly involved in this process but its exact involvement is unknown. To test the working model, it is paramount to investigate where Gcn1 and Gcn2 bind on the ribosome. Ribosomes consist of a large and small subunit, each containing multiple ribosomal proteins placed in unique locations. Identification of ribosomal proteins contacting Gcn1 or Gcn2 will allow for deduction of where Gcn1 and Gcn2 bind on the ribosome. This study aimed to determine Gcn1 and Gcn2 contact points on the large ribosomal subunit, usinga genetic approach. The hypothesis was that if an interaction of Gcn1 or Gcn2 with a particular large ribosomal protein (Rpl) is important for Gcn2 activation, then its overexpression would impair Gcn2 function. Overexpression of several large ribosomal proteins impaired cell growth on a medium triggering amino acid starvation, suggesting Gcn2 activation was impaired. Groups of two or more of these Rpls were found in several regions which contain ribosomal proteins shown or suggested to interact with Gcn1 or Gcn2 previously. This included a region containing the P-stalk proteins (part of the large ribosomal complex) known to contact Gcn2. A region close to the small ribosomal protein Rps10, known to contact Gcn1, was also identified. Another region with Rpls which contacts a protein eEF3, which is suggested to share similar ribosomal contacts as Gcn1, was identified.
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    Discovering links between elongation factors and general amino acid control in Saccharomyces cerevisiae : this thesis is presented in partial fulfilment of the requirements for the degree of Doctor of Philosphy (PhD) in Biochemistry at Massey University, Auckland, New Zealand.
    (Massey University, 2011) Visweswaraiah, Jyothsna
    Continous protein synthesis is essential for life; hence, a steady supply of amino acids must be maintained. In order to respond appropriately to amino acid shortages, cells need to constantly monitor their availability. Cells have a signal transduction pathway, called the general amino acid control (GAAC), for sensing and ameliorating amino acid shortages. Since the sensing occurs on translating ribosomes, the objective of this study was to investigate links between translation elongation and the general amino acid control in S. cerevisiae. In all eukaryotes, Gcn2 and its effector Gcn1 are responsible for monitoring amino acid availability. Active protein synthesis requires eukaryotic translation elongation factors (eEFs) to associate with translating ribosomes. This study focussed on two eEFs, eEF3 and eEF1A, and their potential role in GAAC. Gcn1 has homology to eEF3, which suggests that both proteins utilise overlapping binding sites on the ribosome. Supporting this idea, it was found that over-expression of eEF3 caused sensitivity to amino acid analogues (AAAs), suppressed the growth defect associated with constitutively active Gcn2, and impaired Gcn2 function. The C-terminal domain in eEF3 was found to be responsible for affecting Gcn2 function. Over-expression of this domain was sufficient for ribosome binding and for causing AAAs. These findings suggest that eEF3 influences Gcn1 negatively. For signal perception, Gcn1 and Gcn2 need to access the ribosomal A-site where eEF1A is functional. This suggests a link exists between eEF1A and GAAC. This link was confirmed by the discovery that eEF1A interacts with Gcn2 in vivo. The Gcn2 C-terminal domain was sufficient to precipitate eEF1A, independent of ribosomes, other yeast proteins and RNA. The interaction was lost under amino acid starvation conditions, suggesting that eEF1A is a negative regulator of Gcn2 activation under replete conditions. This study reveals a link between translation elongation and GAAC. As eEF3 and eEF1A are known to interact with each other it is proposed here that they act in concert to inhibit Gcn1 and Gcn2 under replete conditions, hence suggesting a novel mechanism of Gcn2 regulation.