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.
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.