Mechanism of discrimination of natural proteinogenic and nonproteinogenic amino acids in class IA aminoacyl-tRNA synthetases

Aminoacil-tRNA-sintetaze kataliziraju vezanje aminokiselina na 3'-kraj tRNA u reakciji koja se sastoji od dva koraka: prvo dolazi do aktivacije aminokiseline uz pomoć ATP-a pa nastaje aminoacil-adenilat, a zatim do prijenosa aktivirane aminokiseline na 3'- kraj tRNA. Strukturna sličnost aminokiselinskih supstrata i potreba za točnošću reakcije aminoaciliranja uvjetovale su postojanje mehanizama popravka pogrešaka aminoacil-tRNA-sintetaza: popravak se može odvijati prije i poslije prijenosa na tRNA. U ovoj disertaciji istraženi su mehanizmi diskriminacije enzima izoleucil- i valil-tRNA-sintetaze iz bakterije Escherichia coli prema prirodnoj neproteinogenoj aminokiselini norvalinu. IleRS uspješno aktivira norvalin i prenosi ga na tRNAIle, a popravak se vrši prije prijenosa i većim dijelom nakon prijenosa na tRNA. Kao što je to prethodno pokazano za valin, IleRS koristi, uz tRNA-neovisni, i tRNA-ovisni popravak pogreške prije prijenosa za uklanjanje norvalina. Potonji mehanizam je svojstven IleRS i nije dokumentiran kod drugih enzima klase Ia. Norvalin je 3 do 4 puta toksičniji od valina bakteriji E. coli koja eksprimira IleRS s ugašenim popravkom pogreške nakon prijenosa. ValRS slabo aktivira norvalin, pa se već u prvom koraku reakcije aminoaciliranja postiže diskriminacija dovoljna za očuvanje točne biosinteze proteina. Ipak, aktivirani norvalin se efikasno prenosi na tRNAVal, a ako dođe do prijenosa uklanja se popravkom pogreške nakon prijenosa.Aminoacyl-tRNA synthetases catalyze binding of amino acids to the 3'-end of tRNA in a 2-step reaction: the first step is activation of amino acid with ATP to form aminoacyl-adenylate, and in the second step the activated amino acid is transferred to tRNA's 3'-end. Structural similarity of the amino acid substrates and the required accuracy of the aminoacylation reaction directed the development of proofreading mechanisms by aminoacyl-tRNA synthetases: proofreading may occur before or after amino acid transfer to tRNA. In this dissertation, discrimination mechanisms of a natural non-proteinogenic amino acid norvaline by Escherichia coli isoleucyland valyl-tRNA synthetase were explored. IleRS activates norvaline well and transfers it to tRNAIle, and the repair mechanisms may operate prior to or after transfer of norvaline to tRNAIle. As previously shown with valine, besides tRNA-independent, IleRS also exhibits tRNA-dependent pre-transfer editing to eliminate norvaline. The latter mechanism is distinctive for IleRS and its existence has not been proven in other aminoacyl-tRNA synthetases. Norvalin is 3-4 × more toxic than valine to an E. coli strain that contains IleRS with inactivated posttransfer editing. ValRS weakly activates norvaline, and the discrimination achieved in the first step of aminoacylation is high enough to sustain the accuracy of overall protein biosynthesis. Nevertheless, as activated norvaline is efficiently transferred to tRNAVal, ValRS can eliminate norvalyl-tRNAVal through post-transfer editing