Cloning, overexpression and purification of Escherichia coli tRNAIle and tRNAVal

Aminoacil-tRNA-sintetaze su enzimi koji kataliziraju nastajanje kovalentne veze između pripadnog para aminokiseline i molekule transfer tRNA (tRNA). Budući da je točnost aminoaciliranja temeljni preduvjet za vjeran prijenos genetičke informacije, ti enzimi su razvili mehanizme popravka kako bi uklonili pogreške nastale prilikom aktivacije nepripadne aminokiseline (popravak prije prijenosa), odnosno prijenosa pogrešne aminokiseline na tRNA (popravak poslije prijenosa). tRNA vrlo često potiče popravak prije prijenosa aminokiseline ali mehanizam tog procesa još nije poznat. U nekim slučajevima tRNA (npr. tRNAIle) mora sadržavati postranskripcijske modifikacije kako bi mogla poticati reakciju popravka. Velike količine biološki aktivne tRNA potrebne su za istraživanja mehanizma tRNAovisnog popravka izoleucil- i valil-tRNA-sintetaze. Priređeni su rekombinantni plazmidi za prekomjernu ekspresiju tRNAIle i tRNAVal pod kontrolom jakog T7 promotora. Optimiran je sustav za preparativnu proizvodnju molekula tRNA in vivo i razvijena je brza i efikasna metoda pročišćavanja posttranskripcijski modificirane tRNA kromatografijom obrnutih faza. Metodama enzimske kinetike istražena su svojstva pripravljenih tRNA.Aminoacyl-tRNA synthetases (aaRS) are enzymes that covalently join amino acids with their cognate transfer RNAs (tRNAs). As highly accurate aminoacyl-tRNA synthesis is a prerequisite for the precise transmission of genetic information, some aaRSs have developed editing mechanisms to correct initial errors in amino acid activation (pre-transfer) or aminoacyltRNA formation (post-transfer). tRNA often stimulates pre-transfer editing by a yet unknown mechanism. In some cases, tRNA has to be posttranscriptionally modified (tRNAIle for example) in order to stimulate editing. Large amounts of biologically active tRNAs are needed to conduct detailed kinetic studies of tRNA-dependent editing by isoleucyl- and valyl-tRNA synthetases. Recombinant plasmids for in vivo overexpression of tRNAIle and tRNAVal under control of T7 strong promotor have been produced. The overexpression has been optimized for preparative purposes and an efficient purification method by reverse phase chromatography has been developed for purification of posttranscriptionally modified tRNAs. The properties of obtained tRNAs were assesed by enzyme kinetics