Mupirocin sensitivity of isoleucyl-tRNA synthetases from various organisms on protein and cellular level

Izoleucil-tRNA-sintetaza (IleRS) kovalentno veže izoleucin na pripadnu tRNAIle, pri čemu ponekad griješi. Radi toga posjeduje mehanizme popravka vlastite pogreške. Uočena su 2 tipa IleRS koji se strukturno razlikuju u C-terminalnoj domeni. IleRS1 se nalazi kod prokariota, dok se IleRS2 pojavljuje kod eukariota, ali i kod nekih prokariota. IleRS1 je, u pravilu, osjetljiviji na mupirocin od IleRS2 i uz popravak pogreške nakon prijenosa koristi i tRNAovisni popravak misaktivirane aminokiseline, koji nije detektiran na dosad istraživanim IleRS2. U ovom radu želi se preispitati korelacija između osjetljivosti prema mupirocinu i tipa IleRS-a iz više različitih organizama. Uklonirana su 3 gena za IleRS, te je eksprimiran jedan protein, koji će se u daljnjem radu testirati na inhibiciju mupirocinom. Inhibicija rasta bakterijskih stanica (izražena kao minimalna inhibitorna koncentracija (MIC)) mupirocinom ne pokazuje strogo korelaciju s tipom IleRS-a, što može biti posljedica različitog unosa mupirocina u stanice različitih organizama.Isoleucyl-tRNA synthetase (IleRS) covalently binds isoleucine to their cognate tRNAIle, sometimes making mistakes. Therefore, it acquired editing activity to maintain overall accuracy of translation. Two types of IleRS have been observed that differ structurally in the C-terminal domain. IleRS1 is found in prokaryotes, while IleRS2 occurs in eukaryotes as well as in some prokaryotes. Generally, IleRS1 is more sensitive to mupirocin than IleRS2, and in addition to post-transfer editing, uses a tRNA-dependent pretransfer editing, which was not detected on the IleRS2 studied so far. Here we aim to examine the correlation between mupirocin sensitivity and type IleRS from a number of different organisms. In this work, 3 genes coding for IleRS were cloned and one protein was expressed and purified, which will be assayed in vitro for mupirocin inhibition. The inhibition of bacterial cell growth (expressed as minimum inhibitory concentration (MIC)) by mupirocin does not show a strict correlation with the type of IleRS, which may be due to different uptake of mupirocin into cells by different organisms