Aminoacyl-tRNA synthetases are ubiquitous enzymes which are responsible for the fidelity of the translation of the genetic code. The three-dimensional structures of these proteins are important since they enable us to address a number of questions about their function, and to understand how do they recognise the correct amino acid and how can discriminate between various tRNAs of similar shape and size. A comparison between the structures of evolutionary related synthetases can help to answer questions about the way this family of enzymes and the genetic code have coevolved.
E. coli produces two isoforms of Iysyl-tRNA synthetase, a class II aminoacyl-tRNA synthetase: LysS is the constitutive enzyme, while LysU is the product of an inducible gene. The crystal structure of the thermoinducible LysU with Iysine bound to the active site has been determined to 2.8A resolution and compared to other class II synthetases. More work is needed in order to study the reaction mechanism and to analyse the interactions made by ATP, the Iysyl-adenylate intermediate and the tRNA. The structure of the highly homologous LysS was determined in the absence of any substrates.
The project will involve the structure solution and refinement of LysU and LysS in the presence of various substrates and inhibitors, at the highest possible resolution. The structures will then be analysed and compared to identify conformational changes or differences between isoforms of physiological and biochemical relevance. A comparison with other class II synthetases for which crystallographic models of substrates bound to the active sites are known, will also be carried out, in order to determine differences and similarities that rnight have evolutionary implieations. We will use in vitro transcription to produce the tRNALYs transcript and will sereen for erystallisation eonditions with both LysU and LysS. In collaboration with Prof. Blanquet's laboratory (Paris) we will also carry out crystallisation experiments with the overproduced E. coli tRNALYs. Once crystals suitable for X-ray diffraction are obtained, the structure of the tRNA complex will be solved either by molecular replacement or multiple isomorphous replacement, refined and eompared to the uneomplexed enzyme.
Training content (objective, benefit and expected impact)
The accomplishment of this project will give the applicant a theoretical and practical knowledge of all the stages involvec in macromolecular structure determination by single crystal X-ray diffraction, including crystallisation, data collection anc processing, phasing, model building, structure refinement. Italy is currently investing considerable resources intc structural biology, which until very recently has been limited to a very few laboratories. These expertises will therefore bc invaluable to help the expansion of the italian crystallographic community. Links with industry / industrial relevance (22)