Biochemical characterisation of efferent Ire1 signalling

Applications for a competitively-funded Durham Doctoral Studentship in the groups of Dr. Martin Schröder (Department of Biosciences) and Dr. Fernando Dias (Department of Physics) to biochemically characterise production of the endoplasmic reticulum (ER) stress signal by the endoribonuclease activity of Ire1 are invited.
Ire1 is a bifunctional protein kinase endoribonuclease that is activated ER stress caused by the accumulation of un- or misfolded proteins in the ER. Ire1 protects eukaryotic cells from the toxic consequences of elevated levels of unfolded proteins by initiating the processing of the mRNA for the bZIP transcription factor HAC1/XBP1. Cleavage of HAC1 mRNA at two exon-intron junctions by Ire1 followed by ligation of the two HAC1 exons by tRNA ligase removes a translational attenuator from HAC1 mRNA and increases the potency of the transcriptional activation domain of Hac1.
The successful candidate will investigate how accumulation of unfolded proteins in the ER activates the endoribonuclease domain of Ire1. To address this question the successful candidate will (1) characterise binding of adenine nucleotides to wild type Ire1 and several Ire1 mutants, and (2) characterise the effects of nucleotide binding on enzymatic activity and conformation of the endoribonuclease domain. The project will make use of a variety of biochemical and molecular genetic techniques, such as construction and characterisation of the activity of Ire1 mutants in vivo, expression and purification of Ire1 in Escherichia coli, enzyme kinetics, nucleotide binding assays, and fluorescence spectroscopic studies with these purified proteins.