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

Proofreading activities of leucyl-tRNA synthetase prevent noncanonical mistranslation of the Escherichia coli proteome

Sažetak Aminoacil-tRNA-sintetaze (aaRS) su enzimi koji kataliziraju povezivanje aminokiseline i tRNA. Povezivanje isključivo pripadnog para presudno je za točnu biosintezu proteina. AaRS koje nisu dovoljno specifične za pripadnu aminokiselinu imaju složene mehanizme popravka vlastitih pogrešaka. Popravak se može odvijati prije (hidroliza aminoacil-adenilata) ili nakon prijenosa aminokiseline na tRNA (hidroliza misacilirane tRNA). U ovoj disertaciji detaljno su okarakterizirane sintetska i hidrolitičke reakcije leucil-tRNA-sintetaze (LeuRS) iz bakterije E. coli. Pokazano je da LeuRS ne koristi tRNA-ovisan popravak pogreške prije prijenosa i da pogreške uklanja kroz hidrolizu misacilirane tRNA. Izoleucin je u literaturi istaknut kao dobar supstrat LeuRS koji ugrožava točnost dekodiranja leucinskih kodona. Kinetički eksperimenti pokazuju da LeuRS u sintetskoj reakciji učinkovito isključuje izoleucin, zbog čega popravak pogreške nema ulogu u sprječavanju mistranslacije leucinskih kodona izoleucinom. Nedavno je pokazano da se norvalin, neproteinogena aminokiselina, nakuplja u bakterijskoj citoplazmi u uvjetima nedostatka kisika. Rezultati ove disertacije pokazuju da je glavna biološka funkcija hidrolitičkog popravka LeuRS isključivanje norvalina iz genetičkog koda. Kroz ovo istraživanje otkrivena je poveznica popravka pogreške LeuRS i mehanizama prilagodbe bakterija na rast u uvjetima sa smanjenom količinom kisika.Aminoacyl-tRNA synthetases (aaRS) are enzymes that catalyse pairing of amino acids and tRNAs. As coupling of cognate pairs is crucial for accurate protein biosynthesis, aaRSs have developed complex proofreading mechanisms for correction of errors made in amino acid selection. Proofreading may occur before (hydrolysis of aminoacyl-adenylate), or after transfer of amino acid to tRNA (hydrolysis of misacylated tRNA). Synthetic and hydrolytic reactions of leucyl-tRNA synthetase (LeuRS) are characterised in detail within this thesis. The results demonstrate that LeuRS lacks tRNA-dependent pre-transfer editing and relies on robust post-transfer editing to clear aminoacylation errors. Isoleucine is generally thought to be a significant threat for accurate leucylation. However, kinetic experiments demonstrate that LeuRS efficiently excludes isoleucine in the synthetic reaction and that hydrolytic repair does not have a role in isoleucine elimination. A recent study showed that norvaline, a non-proteinogenic amino acid, accumulates in bacterial cytoplasm during oxygen deprivation. It appears that the major biological role of LeuRS editing is exclusion of norvaline from the genetic code. Research within this thesis reveals that LeuRS editing is interconnected with cellular mechanisms that ensure bacterial adaptability to low-oxygen environment

Efficiently Activated Serine Analogue is Not Transferred to Yeast tRNASer

Covalent attachment of cognate amino acid to the cognate tRNA is a prerequisite for the faithful synthesis of proteins in the cell. Aminoacylation of tRNA, catalyzed by aminoacyl-tRNA synthetases (aaRSs), proceeds by a two-step reaction whereby amino acid is first activated and then transferred to the 3\u27-ribose of tRNA. Serine hydroxamate (SerHX) is an interesting analogue of serine as it exhibits antimicrobial activity due to its inhibition of serylation in yeast and Escherichia coli. SerHX also mimics a noncognate substrate of yeast seryl-tRNA synthetase (ScSerRS) since it is efficiently activated and edited by this enzyme. However, whether this analogue is also transferred to tRNA during the second step of aminoacylation was not previously known. Here we show, for the first time, that aminoacylation of yeast tRNA with SerHX does not occur at a measurable rate, suggesting that the transfer is less tolerable toward SerHX than the activation step

Austria – Vienna – Blick vom Hochhaus auf St. Stephan

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Lack of Discrimination Against Non-proteinogenic Amino Acid Norvaline by Elongation Factor Tu from Escherichia coli

The GTP-bound form of elongation factor Tu (EF-Tu) brings aminoacylated tRNAs (aa-tRNA) to the A-site of the ribosome. EF-Tu binds all cognate elongator aa-tRNAs with highly similar affinities, and its weaker or tighter binding of misacylated tRNAs may discourage their participation in translation. Norvaline (Nva) is a non-proteinogenic amino acid that is activated and transferred to tRNALeu by leucyltRNA synthetase (LeuRS). No notable accumulation of Nva-tRNALeu has been observed in vitro, because of the efficient post-transfer hydrolytic editing activity of LeuRS. However, incorporation of norvaline into proteins in place of leucine does occur under certain conditions in vivo. Here we show that EF-Tu binds Nva-tRNALeu and Leu-tRNALeu with similar affinities, and that Nva-tRNALeu and Leu-tRNALeu dissociate from EF-Tu at comparable rates. The inability of EF-Tu to discriminate against norvaline may have driven evolution of highly efficient LeuRS editing as the main quality control mechanism against misincorporation of norvaline into proteins. (doi: 10.5562/cca2173

Efficiently Activated Serine Analogue is Not Transferred to Yeast tRNASer

Covalent attachment of cognate amino acid to the cognate tRNA is a prerequisite for the faithful synthesis of proteins in the cell. Aminoacylation of tRNA, catalyzed by aminoacyl-tRNA synthetases (aaRSs), proceeds by a two-step reaction whereby amino acid is first activated and then transferred to the 3\u27-ribose of tRNA. Serine hydroxamate (SerHX) is an interesting analogue of serine as it exhibits antimicrobial activity due to its inhibition of serylation in yeast and Escherichia coli. SerHX also mimics a noncognate substrate of yeast seryl-tRNA synthetase (ScSerRS) since it is efficiently activated and edited by this enzyme. However, whether this analogue is also transferred to tRNA during the second step of aminoacylation was not previously known. Here we show, for the first time, that aminoacylation of yeast tRNA with SerHX does not occur at a measurable rate, suggesting that the transfer is less tolerable toward SerHX than the activation step