Nonorthologous replacement of lysyl-tRNA synthetase prevents addition of lysine analogues to the genetic code

Abstract

Insertion of lysine during protein synthesis depends on the enzyme lysyl-tRNA synthetase (LysRS), which exists in two unrelated forms, LysRS1 and LysRS2. LysRS1 has been found in most archaea and some bacteria, and LysRS2 has been found in eukarya, most bacteria, and a few archaea, but the two proteins are almost never found together in a single organism. Comparison of structures of LysRS1 and LysRS2 complexed with lysine suggested significant differences in their potential to bind lysine analogues with backbone replacements. One such naturally occurring compound, the metabolic intermediate S-(2-aminoethyl)-L-cysteine, is a bactericidal agent incorporated during protein synthesis via LysRS2. In vitro tests showed that S-(2-aminoethyl)-L-cysteine is a poor substrate for LysRS1, and that it inhibits LysRS1 200-fold less effectively than it inhibits LysRS2. In vivo inhibition by S-(2-aminoethyl)-L-cysteine was investigated by replacing the endogenous LysRS2 of Bacillus subtilis with LysRS1 from the Lyme disease pathogen Borrelia burgdorferi. B. subtilis strains producing LysRS1 alone were relatively insensitive to growth inhibition by S-(2-aminoethyl)-L-cysteine, whereas a WT strain or merodiploid strains producing both LysRS1 and LysRS2 showed significant growth inhibition under the same conditions. These growth effects arising from differences in amino acid recognition could contribute to the distribution of LysRS1 and LysRS2 in different organisms. More broadly, these data demonstrate how diversity of the aminoacyltRNA synthetases prevents infiltration of the genetic code by noncanonical amino acids, thereby providing a natural reservoir of potential antibiotic resistance.

Downward causation by information control in micro-organisms

“…They establish in vivo the existence of various classes of functional equivalence within the OS genetic network [22][23][24] and the metabolic networks [52,53] in bacteria, archaea and lower eukaryotes (e.g. yeast [53]).…”

“…(b) (i) LysyltRNA synthetases (LysRS) have been found in different organisms and are categorized as either class I or class II. Substituting a class II bacterial LysRS by a class I archaeal LysRS still allows the bacteria to operate the COS [22].…”

A natural genetic code expansion cassette enables transmissible biosynthesis and genetic encoding of pyrrolysine

“…Assays were carried out in the presence of the metabolites extracted from the equivalent of 10 mg of dry weight of E. coli. Aminoacylation of the tRNA Pyl in the extracted tRNA pool of M. acetivorans was carried out and detected by Northern blotting of acid-urea gels, as described (9,40). Pyrophosphate exchange assays also follow previously described protocols (9,41), except that 5.5 M PylS in 100 l of total assay volume was used, and 20 l of aliquots was taken at each time point.…”

Aminoacyl-tRNAs: setting the limits of the genetic code

“…More recently it has become clear that amino acid specificity is further enhanced by the existence of a natural reservoir of diverse synthetase alleles. This pool of synthetases, with slight differences in specificity towards molecules outside the canonical set of amino acids, serves to both exclude amino acid analogs from translation (Jester et al 2003) and provide resistance against inhibitory amino acid mimics (Brown et al 2003).…”