RESUMO
Genetic code expansion technologies supplement the natural codon repertoire with assignable variants in vivo, but are often limited by heterologous translational components and low suppression efficiencies. Here, we explore engineered Escherichia coli tRNAs supporting quadruplet codon translation by first developing a library-cross-library selection to nominate quadruplet codon-anticodon pairs. We extend our findings using a phage-assisted continuous evolution strategy for quadruplet-decoding tRNA evolution (qtRNA-PACE) that improved quadruplet codon translation efficiencies up to 80-fold. Evolved qtRNAs appear to maintain codon-anticodon base pairing, are typically aminoacylated by their cognate tRNA synthetases, and enable processive translation of adjacent quadruplet codons. Using these components, we showcase the multiplexed decoding of up to four unique quadruplet codons by their corresponding qtRNAs in a single reporter. Cumulatively, our findings highlight how E. coli tRNAs can be engineered, evolved, and combined to decode quadruplet codons, portending future developments towards an exclusively quadruplet codon translation system.
Assuntos
Anticódon/metabolismo , Códon/metabolismo , Evolução Molecular Direcionada , Escherichia coli/genética , RNA de Transferência/genética , Aminoácidos/genética , Aminoacil-tRNA Sintetases/metabolismo , Clonagem Molecular , Escherichia coli/enzimologia , Proteínas de Escherichia coli/biossíntese , Biossíntese de Proteínas , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA de Transferência/metabolismoRESUMO
5-Methylaminomethyl-2-selenouridine (mnm(5)Se(2)U) is found in the first position of the anticodon in certain tRNAs from bacteria, archaea and eukaryotes. This selenonucleoside is formed in Escherichia coli from the corresponding thionucleoside mnm(5)S(2)U by the monomeric enzyme YbbB. This nucleoside is present in the tRNA of Methanococcales, yet the corresponding 2-selenouridine synthase is unknown in archaea and eukaryotes. Here we report that a bipartite ybbB ortholog is present in all members of the Methanococcales. Gene deletions in Methanococcus maripaludis and in vitro activity assays confirm that the two proteins act in trans to form in tRNA a selenonucleoside, presumably mnm(5)Se(2)U. Phylogenetic data suggest a primal origin of seleno-modified tRNAs.
Assuntos
Proteínas Arqueais/metabolismo , Mathanococcus/genética , Compostos Organosselênicos/metabolismo , RNA de Transferência/metabolismo , Selênio/metabolismo , Tiossulfato Sulfurtransferase/metabolismo , Uridina/análogos & derivados , Sequência de Aminoácidos , Animais , Proteínas Arqueais/química , Proteínas Arqueais/classificação , Proteínas Arqueais/genética , Ligases/química , Ligases/classificação , Ligases/genética , Ligases/metabolismo , Mathanococcus/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Compostos Organosselênicos/química , Filogenia , Estrutura Terciária de Proteína , RNA de Transferência/classificação , RNA de Transferência/genética , Alinhamento de Sequência , Tiossulfato Sulfurtransferase/química , Tiossulfato Sulfurtransferase/genética , Uridina/química , Uridina/genética , Uridina/metabolismoRESUMO
The methanogenic archaea Methanococcus jannaschii and M. maripaludis contain an atypical seryl-tRNA synthetase (SerRS), which recognizes eukaryotic and bacterial tRNAsSer, in addition to the homologous tRNASer and tRNASec species. The relative flexibility in tRNA recognition displayed by methanogenic SerRSs, shown by aminoacylation and gel mobility shift assays, indicates the conservation of some serine determinants in all three domains. The complex of M. maripaludis SerRS with the homologues tRNASer was isolated by gel filtration chromatography. Complex formation strongly depends on the conformation of tRNA. Therefore, the renaturation conditions for in vitro transcribed tRNASer(GCU) isoacceptor were studied carefully. This tRNA, unlike many other tRNAs, is prone to dimerization, possibly due to several stretches of complementary oligonucleotides within its sequence. Dimerization is facilitated by increased tRNA concentration and can be diminished by fast renaturation in the presence of 5 mm magnesium chloride.