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.

Abstract

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.