Purification and crystallization of RelA Mutants

Abstract

The bacterial stringent response is the mechanism that bacteria have to respond to stress conditions such as limiting amino acids, limiting phosphate or fatty acids, carbon limitation or heat shock. When this happens, the stringent response is activated by the small alarmone (p)ppGpp. The accumulation of this compound causes two main situations: the bacterial cellular activity is mostly turned off and the expression systems are focused in expressing survival components. (p)ppGpp is synthesised by RelA enzyme, which catalyses the reaction between GTP or GDP and ATP in order to form (p)ppGpp. This guanosine tetra or pentaphosphate, activates the pathway to modify the action of RNA polymerase and the cellular expression systems. RelA has 5 domains: The N-terminal Hydrolase, Synthetase, ThrRS, GTPase and SpoT domain (TGS), Zinc-finger domain and RNA recognition motif. In a recent study (Brown, Fernández, Gordiyenko, & Ramakrishnan, 2016), the ribosome-bound structure has been revealed, and it shows that RelA wraps around the ribosomal A-site with the last two C-terminal domains, although it has some flexible parts which remain unknown. The present study aimed to purify and crystallize RelA and RelA mutants. Purification and crystallization protocols for RelA and two RelA mutants (RelA Δ2 and RelA Δ5) are presented. For RelA Δ2, the purified protein was obtained by changing the cell line and using different FPLC (Fast Protein Liquid Chromatography) systems. For RelA Δ5, the same purification strategy was followed, and lead to a high amount and pure protein. Crystallization trays were set for both proteins, but no significant results were shown. In RelA-G-His (full construction of the protein), a different strategy was followed. The plan has been to form a complex between a nanobody and RelA to stabilize RelA, thus making the crystallization possible, but for a matter of time it could not be completed. Despite this, a new approach for future crystallization attempts is presented, because the complex was successfully bound using the two purified proteins