Properties of the lysyl-tRNA synthetase gene and product from the extreme thermophile Thermus thermophilus

Abstract

A DNA region carrying lysS, the gene encoding the lysyl-tRNA synthetase, was cloned from the extreme thermophile prokaryote Thermus thermophilus VK-1 and sequenced. The Recently, the availability of the primary sequences of all 20 aminoacyl-tRNA synthetases (aaRSs) and the three-dimensional structures of a few of them allowed the determination of resemblances among subsets of these enzymes and, finally, the partition of the 20 aaRSs into two classes (10, 13). The first class is characterized by the presence of two consensus peptidic motifs (22,23,47) and of a nucleotide-binding fold (Rossman fold) (40). These features are not found in the class II aaRSs, which, in contrast, are built around an antiparallel p-sheet core (10, 37) and display three different consensus motifs (13). This classification is further sustained by the observation that class I aaRSs aminoacylate tRNA on the 2'-OH group of the 3'-terminal ribose, while class II aaRSs (with the exception of phenylalanyl-tRNA synthetase) esterify the 3'-OH group (13).Among the class II enzymes, aspartyl-, lysyl-, and asparaginyl-tRNA synthetases form a group with particularly close sequence homology (2, 14, 32). For instance, Escherichia coli lysyl-tRNA synthetase (LysRS), encoded by two distinct genes, lysS and lysU (32), has 19, 23, and 20% identity with the aspartyl-tRNA synthetases from E. coli, Saccharomyces cerevisiae, and rat, respectively. Recently, the three-dimensional structure of S. cerevisiae AspRS complexed with tRNAAP became available (6, 41), and the structures of the AspRS from E. coli and Thermus thermophilus are currently being determined (12,39). This opens the possibility of comparing AspRS and LysRS at the structural level and understanding the basis of the amino acid specificities of these highly related enzymes.In this study, isolation of the T thermophilus LysRS gene was undertaken with the aim of determining the structure of this synthetase. The nucleotide sequence of this gene, lysS, was determined. Both overproduction from E. coli and purification of an active form of the highly thermostable Thermus LysRS were achieved. In addition, we show that, unexpectedly, Ther-* Corresponding author.

Archaeal-type lysyl-tRNA synthetase in the Lyme disease spirochete Borrelia burgdorferi

“…A comparable ORF was found in the genome of another spirochete, T. pallidum (Fig. 2, Table 1), although it should be noted that in this case a sequence was also detected that encodes a 345-amino acid protein similar (32% identity and 52% similarity at the amino acid level) to the carboxylterminal 70% of the class II-type LysRS of Thermus thermophilus (27).…”

Genetic code origins: Experiments confirm phylogenetic predictions and may explain a puzzle

“…Thus, at least one lysyl-tRNA synthetase appeared after tRNA Lys was established (17). Can charging of the same tRNA For the in vivo experiments, they take advantage of a strain of Escherichia coli that is deficient in lysyl-tRNA synthetase (22). As a result of this deficiency, the cells cannot grow unless provided with an active lysyl-tRNA synthetase.…”

An expanded genetic code with a functional quadruplet codon

“…Genes for PhKRS⌬ and EcKRS under the constitutive glnRS promoter of the pGLN expression vector were used to transform temperature-sensitive E. coli cells PAL⌬S⌬UTR(pMAKlysU) (30). The deletion strain PAL⌬S⌬UTR is deficient in lysyl-tRNA synthetase because of lysS͞lysU double mutations; pMAKlysU is a temperaturesensitive plasmid that encodes lysU and complements the lysS͞ lysU mutations.…”