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Durham e-Theses
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Intragenic suppression of RNase-defective point
mutation of the catalytic aspartate in the protein
kinase domain of Ire1

OBIDAN, AMNAH,MAHMOUD (2023) Intragenic suppression of RNase-defective point
mutation of the catalytic aspartate in the protein
kinase domain of Ire1.
Doctoral thesis, Durham University.

Full text not available from this repository.
Author-imposed embargo until 06 December 2024.


In eukaryotic cells, proper folding of secretory and transmembrane proteins occurs within the
endoplasmic reticulum (ER) before their exit the ER. The accumulation of unfolded proteins
activates a response known as the unfolded protein response (UPR), mediated by Ire1. In
Saccharomyces cerevisiae, Ire1 activation leads to the splicing of HAC1 mRNA, which
encodes a transcription factor involved in the UPR. In this study, we focused on specific
mutations within the protein kinase domain of Ire1. Specifically, the protein kinase domain
was subjected to mutations to alter the catalytic aspartate D797 and lysine K799, which
interacts with the terminal phosphate group of ATP, to alanine. Also, point mutations in the
Mg2+ coordinating loop converted asparagine N802 and aspartic acid D828 to alanine. To
investigate the impact of these mutations, we performed several experiments. Northern blot
analysis was employed to detect the splicing of HAC1 mRNA, as it serves as an indicator of
Ire1 activity and UPR induction. Additionally, β-galactosidase reporter assays were
conducted to assess the expression of a UPRE-lacZ reporter gene, which is also regulated by
the Ire1-Hac1 signalling pathway. The results demonstrated that single mutations in the
catalytic domain and Mg2+ coordinating loop (K799A, D797A, N802A, and D828A) led to
decreased levels of HAC1 mRNA and reduced expression of the UPRE-lacZ reporter gene
compared to the WT Ire1. Furthermore, the D797A mutant strain exhibited decreased
survival under ER stress conditions when compared to other mutants within the Mg2+
coordinating loop and catalytic domain. Interestingly, the D797A mutation resulted in lower
levels of HAC1 mRNA species and β-galactosidase activity. However, introducing additional
mutations such as D797A N802A or D797A K799A N802A led to significant increases in βgalactosidase activity, the percentage of HAC1 mRNA, and restored growth compared to the
single D797A Ire1 mutant. Notably, the expression levels of WT and protein kinase mutants
were similar. In conclusion, the findings suggest that introducing specific additional
mutations, such as K799A Ire1, D828A Ire1, or N802A Ire1, to the single D797A Ire1 mutant
can restore the signalling activity of Ire1.

Item Type:Thesis (Doctoral)
Award:Doctor of Philosophy
Keywords:IRE1, Intragenic suppression and RNA
Faculty and Department:Faculty of Science > Biological and Biomedical Sciences, School of
Thesis Date:2023
Copyright:Copyright of this thesis is held by the author
Deposited On:07 Dec 2023 10:20

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