Benfang Ruan

Quality control despite mistranslation caused by an ambiguous genetic code

A high level of accuracy during protein synthesis is considered essential for life. Aminoacyl-tRNA synthetases (aaRSs) translate the genetic code by ensuring the correct pairing of amino acids with their cognate tRNAs. Because some aaRSs also produce misacylated aminoacyl-tRNA (aa-tRNA) in vivo, we addressed the question of protein quality within the context of missense suppression by Cys-tRNAPro, Ser-tRNAThr, Glu-tRNAGln, and Asp-tRNAAsn. Suppression of an active-site missense mutation leads to a mixture of inactive mutant protein (from translation with correctly acylated aa-tRNA) and active enzyme indistinguishable from the wild-type protein (from translation with misacylated aa-tRNA). Here, we provide genetic and biochemical evidence that under selective pressure, Escherichia coli not only tolerates the presence of misacylated aa-tRNA, but can even require it for growth. Furthermore, by using mass spectrometry of a reporter protein not subject to selection, we show that E. coli can survive the ambiguous genetic code imposed by misacylated aa-tRNA tolerating up to 10% of mismade protein. The editing function of aaRSs to hydrolyze misacylated aa-tRNA is not essential for survival, and the EF-Tu barrier against misacylated aa-tRNA is not absolute. Rather, E. coli copes with mistranslation by triggering the heat shock response that stimulates nonoptimized polypeptides to achieve a native conformation or to be degraded. In this way, E. coli ensures the presence of sufficient functional protein albeit at a considerable energetic cost.

Activation of the Pyrrolysine Suppressor tRNA Requires Formation of a Ternary Complex with Class I and Class II Lysyl-tRNA Synthetases

Monomethylamine methyltransferase of the archaeon Methanosarcina barkeri contains a rare amino acid, pyrrolysine, encoded by the termination codon UAG. Translation of this UAG requires the aminoacylation of the corresponding amber suppressor tRNAPyl. Previous studies reported that tRNAPyl could be aminoacylated by the synthetase-like protein PylS. We now show that tRNAPyl is efficiently aminoacylated in the presence of both the class I LysRS and class II LysRS of M. barkeri, but not by either enzyme acting alone or by PylS. In vitro studies show that both the class I and II LysRS enzymes must bind tRNAPyl in order for the aminoacylation reaction to proceed. Structural modeling and selective inhibition experiments indicate that the class I and II LysRSs form a ternary complex with tRNAPyl, with the aminoacylation activity residing in the class II enzyme.

Protein Synthesis in Escherichia coli with Mischarged tRNA

Two types of aspartyl-tRNA synthetase exist: the discriminating enzyme (D-AspRS) forms only Asp-tRNA(Asp), while the nondiscriminating one (ND-AspRS) also synthesizes Asp-tRNA(Asn), a required intermediate in protein synthesis in many organisms (but not in Escherichia coli). On the basis of the E. coli trpA34 missense mutant transformed with heterologous ND-aspS genes, we developed a system with which to measure the in vivo formation of Asp-tRNA(Asn) and its acceptance by elongation factor EF-Tu. While large amounts of Asp-tRNA(Asn) are detrimental to E. coli, smaller amounts support protein synthesis and allow the formation of up to 38% of the wild-type level of missense-suppressed tryptophan synthetase.

Methanocaldococcus jannaschii Prolyl-tRNA Synthetase Charges tRNAPro with Cysteine

Methanocaldococcus jannaschiiprolyl-tRNA synthetase (ProRS) was previously reported to also catalyze the synthesis of cysteinyl-tRNACys(Cys-tRNACys) to make up for the absence of the canonical cysteinyl-tRNA synthetase in this organism (Stathopoulos, C., Li, T., Longman, R., Vothknecht, U. C., Becker, H., Ibba, M., and Söll, D. (2000) Science 287, 479–482; Lipman, R. S., Sowers, K. R., and Hou, Y. M. (2000)Biochemistry 39, 7792–7798). Here we show by acid urea gel electrophoresis that pure heterologously expressed recombinantM. jannaschii ProRS misaminoacylates M. jannaschii tRNAPro with cysteine. The enzyme is unable to aminoacylate purified mature M. jannaschiitRNACys with cysteine in contrast to facile aminoacylation of the same tRNA with cysteine by Methanococcus maripaludiscysteinyl-tRNA synthetase. Although M. jannaschii ProRS catalyzes the synthesis of Cys-tRNAPro readily, the enzyme is unable to edit this misaminoacylated tRNA. We discuss the implications of these results on the in vivo activity of the M. jannaschii ProRS and on the nature of the enzyme involved in the synthesis of Cys-tRNACys in M. jannaschii.

Cys-tRNACys formation and cysteine biosynthesis in methanogenic archaea: two faces of the same problem?

Abstract.Aminoacyl-tRNA (transfer RNA) synthetases are essential components of the cellular translation machinery as they provide the ribosome with aminoacyl-tRNAs. Aminoacyl-tRNA synthesis is generally well understood. However, the mechanism of Cys-tRNACys formation in three methanogenic archaea (Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus and Methanopyrus kandleri) is still unknown, since no recognizable gene for a canonical cysteinyl-tRNA synthetase could be identified in the genome sequences of these organisms. Here we review the different routes recently proposed for Cys-tRNACys formation and discuss its possible link with cysteine biosynthesis in these methanogenic archaea.

Cysteinyl-tRNA formation and prolyl-tRNA synthetase.

Aminoacyl‐tRNA (AA‐tRNA) formation is a key step in protein biosynthesis. This reaction is catalyzed with remarkable accuracy by the AA‐tRNA synthetases, a family of 20 evolutionarily conserved enzymes. The lack of cysteinyl‐tRNA (Cys‐tRNA) synthetase in some archaea gave rise to the discovery of the archaeal prolyl‐tRNA (Pro‐tRNA) synthetase, an enzyme capable of synthesizing Pro‐tRNA and Cys‐tRNA. Here we review our current knowledge of this fascinating process.