Identification of ribosomal proteins that are necessary for fully activating the protein kinase Gcn2 : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Albany, New Zealand
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Date
2014
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Massey University
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Abstract
The environment in which cells grow often changes rapidly and in order to survive,
cells need to adjust their metabolic pathway to these changes. Vitally important for
all organisms is the constant availability of amino acids as they are building blocks
for proteins. Proteins are essential molecules involved in most biological processes in
a cell. Yeast and mammals overcome amino acid limitation by switching on a
signalling pathway named General Amino Acid Control (GAAC), which triggers a
decrease in general protein synthesis by inhibiting translation initiation while
upregulating the transcription of stress-response genes.
For sensing starvation in yeast, the GAAC requires the kinase Gcn2 and its effector
protein Gcn1. Gcn2 phosphorylates the α-subunit of the eukaryotic initiation factor 2
(eIF2α), which ultimately induces the selective expression of stress-response genes,
leading to the de novo synthesis of all amino acids. In order to recognize the
deacylated tRNA as an immediate signal for starvation, Gcn1 and Gcn2 need to be in
direct contact and associated with the translating ribosome. The current model for
sensing starvation by Gcn2 suggests that deacylated tRNA enters the ribosomal Asite
and Gcn1 concomitantly transfers the starvation signal to Gcn2. However, the
molecular details of this process are still unclear. Deletion analysis of GCN1,
suggested that Gcn1 has multiple contact points with the ribosome. We therefore aim
to uncover ribosomal proteins that are required to fully activate Gcn2 in order to
better understand the starvation recognition process. The fact that Gcn1 has many
ribosomal contact points implies that the deletion of one contact point will not
remove Gcn1 from the ribosome and therefore maintains Gcn2 activation. This
allows us to identify Gcn1-ribosome interaction points which are not only required to
position Gcn1on the ribosome but also facilitate in Gcn1 mediated Gcn2 activation
per se.
Genetic studies conducted in this thesis reveal that ribosomal proteins rps18, rps26,
rps28, rpl21 and rpl34 are necessary for full Gcn2 activation. The deletion of their
genes resulted in an impaired growth on starvation media and in a reduction in eIF2α
phosphorylation. With these results we are able to create a first map of Gcn1 contact
points of the ribosome that are necessary to promote Gcn2 activation. Two ribosomal
proteins that are necessary for fully activated Gcn2 are located on the large ribosomal
subunit. Three others are located on the ribosomal head region of the small ribosomal
subunit in proximity to the A-site region. Considering that Gcn1 is a large protein,
our results support the idea that Gcn1 has multiple contact points with the ribosome
and that some important contact points for Gcn2 activation are located near the
ribosomal A-site.
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Keywords
Amino acids, Ribosomal proteins, Protein synthesis, General Amino Acid Control (GAAC), Protein kinase, Gcn2, Research Subject Categories::NATURAL SCIENCES::Chemistry::Biochemistry