The previously described screens investigated whether the ribosomal protein knockdown affects the activation of Gcn2 and therefore the cell’s ability to overcome amino acid deprivation. We know that the direct contact of Gcn1 as well as Gcn2 to the ribosome is crucial for Gcn2 activation. As Gcn2 location on the ribosome is not fully known, it is possible that the observed SM sensitivity of some strains could result from the loss of ribosomal contact points of either Gcn1 or Gcn2. It was therefore necessary to uncover which of the ribosomal proteins contact Gcn1 or Gcn2. To address this task we aimed to overexpress Gcn1 or Gcn2 in the rpx∆ strains. We expected that the overexpression of Gcn1 or Gcn2 will strengthen the binding of Gcn1 or Gcn2 to the ribosome based on mass action. As a result the SMS phenotype of the deletion strain would be rescued. If Gcn2 was able to rescue the phenotype when overexpressed in a rpx∆ strain, it would suggest that the deleted ribosomal protein was a binding site for Gcn2. The same is applicable for the overexpression of Gcn1.
For this purpose our aim was to subject the five rpx∆ strains, which had impaired Gcn2 activation as, judged by reduced eIF2α-P to a suppression assay. We therefore transformed the strains with a high copy (hc) plasmid expressing myc-tagged GCN1 or GCN2 from their own promoter. The same transformation was done with the control WT strains BY4741 and BY4742 and gcn1∆ strain. All strains have additionally been transformed with an empty vector control. The strains were then subjected to a semi- quantitative growth assay following the same procedure as in chapter 3. Due to time restrictions, we chose to test the feasibility of this approach with rps18A∆ and rpl21A∆
strain. We decided on transforming high copy GCN1 into rps18A∆ strain and high copy GCN2 into rpl21A∆ strain as we expected that rps18 will be a binding partner of Gcn1, due to its location on the 40S head region, whereas Gcn2 was shown to bind the 60S
Identification of ribosomal binding sites of Gcn1 that are necessary to promote Gcn2 activity
subunit (Ramirez et al., 1991), hence it could possibly bind rpl21. If Gcn1 requires the binding of rps18 to activate Gcn2, then the overexpression of Gcn1 in rps18A∆ should rescue the SM sensitivity phenotype of the strain. If Gcn2 needs to bind rps21, then the overexpression of Gcn2 in rpl21A∆ should rescue the SM sensitivity phenotype of the strain.
Figure 17: Suppression assay of rps18A∆ strain with high copy (hc) Gcn1.
As expected, the transformed WT strains did grow under all conditions and did not show a difference in growth between WT containing the plasmid or the empty vector (Figure 17). Our negative control gcn1∆ strain containing empty vector did not grow under amino acid starvation, but grew when it contained high copy GCN1 instead, suggesting that the drug triggered the amino acid starvation and that high copy Gcn1 can rescue the Gcn- phenotype of the gcn1∆ strain. Furthermore it can be observed, that the rps18A∆ strain with empty vector still has a similar SM sensitivity phenotype as found in Figure 8. The strain containing high copy GCN1 plasmid however grew similar to that containing empty vector under amino acid starvation, suggesting that the hcGcn1 plasmid was unable to rescue the weak SM sensitivity phenotype of the strain (Figure 17), for more see chapter 4.1.
Identification of ribosomal binding sites of Gcn1 that are necessary to promote Gcn2 activity
The control strains in Figure 18 showed expected behaviour. The WT strains did grow under all conditions and did not show a difference in growth between WT containing the plasmid or the empty vector. Also as expected, the gcn2∆ strain containing empty vector did not grow under amino acid starvation, but grew when it contained high copy GCN2 instead, indicating that the starvation was induced and that high copy Gcn2 can rescue the phenotype of the strain. We observed, that the rpl21A∆ strain transformed with empty vector maintained its SM sensitivity phenotype (chapter 3.1, Figure 9) and that rpl21A∆ strain containing high copy GCN2 showed a similar phenotype. This suggested that high copy number of Gcn2 cannot rescue the phenotype caused by RPL21A deletion.
Discussion
4.
Discussion
Parts of the results described in this chapter were presented in the following international and national conferences:
National conferences:
• QMB – Queenstown Molecular Biology conference 2013 “ Mapping Gcn1 on the ribosome” by Viviane Jochmann (Poster presentation, won the best poster award)
• INMS – Institute of Natural and Mathematical Sciences student conference 2013 “ Mapping Gcn1 on the ribosome” by Viviane Jochmann (Poster presentation)
• YPD –Yeast, Products, Discovery Meeting 2013
“Mapping Gcn1 on the ribosome” by Viviane Jochmann (Poster presentation)
• NZMS –New Zealand Microbiological Society conference 2013, New Zealand “Gcn1 binding to the 40S ribosome head region is essential for fully activating the ancient nutrient-sensor-kinase Gcn2”. (Oral presentation by E. Sattlegger) International conferences:
• EMBL – European Molecular Biology Laboratory conference 2013, Germany. ”Gcn1 binding to the 40S ribosome head region is essential for fully activating eIF2α kinase Gcn2” (Oral presentation by E. Sattlegger)
Discussion