Debra Tumbula-Hansen

Transfer RNA-dependent amino acid biosynthesis: An essential route to asparagine formation

Biochemical experiments and genomic sequence analysis showed that Deinococcus radiodurans and Thermus thermophilus do not possess asparagine synthetase (encoded by asnA or asnB), the enzyme forming asparagine from aspartate. Instead these organisms derive asparagine from asparaginyl-tRNA, which is made from aspartate in the tRNA-dependent transamidation pathway [Becker, H. D. & Kern, D. (1998) Proc. Natl. Acad. Sci. USA 95, 12832–12837; and Curnow, A. W., Tumbula, D. L., Pelaschier, J. T., Min, B. & Söll, D. (1998) Proc. Natl. Acad. Sci. USA 95, 12838–12843]. A genetic knockout disrupting this pathway deprives D. radiodurans of the ability to synthesize asparagine and confers asparagine auxotrophy. The organisms capacity to make asparagine could be restored by transformation with Escherichia coli asnB. This result demonstrates that in Deinococcus, the only route to asparagine is via asparaginyl-tRNA. Analysis of the completed genomes of many bacteria reveal that, barring the existence of an unknown pathway of asparagine biosynthesis, a wide spectrum of bacteria rely on the tRNA-dependent transamidation pathway as the sole route to asparagine.

Coevolution of an aminoacyl-tRNA synthetase with its tRNA substrates

Glutamyl-tRNA synthetases (GluRSs) occur in two types, the discriminating and the nondiscriminating enzymes. They differ in their choice of substrates and use either tRNAGlu or both tRNAGlu and tRNAGln. Although most organisms encode only one GluRS, a number of bacteria encode two different GluRS proteins; yet, the tRNA specificity of these enzymes and the reason for such gene duplications are unknown. A database search revealed duplicated GluRS genes in >20 bacterial species, suggesting that this phenomenon is not unusual in the bacterial domain. To determine the tRNA preferences of GluRS, we chose the duplicated enzyme sets from Helicobacter pylori and Acidithiobacillus ferrooxidans. H. pylori contains one tRNAGlu and one tRNAGln species, whereas A. ferrooxidans possesses two of each. We show that the duplicated GluRS proteins are enzyme pairs with complementary tRNA specificities. The H. pylori GluRS1 acylated only tRNAGlu, whereas GluRS2 was specific solely for tRNAGln. The A. ferrooxidans GluRS2 preferentially charged \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{tRNA}}_{{\mathrm{UUG}}}^{{\mathrm{Gln}}}\end{equation*}\end{document}. Conversely, A. ferrooxidans GluRS1 glutamylated both tRNAGlu isoacceptors and the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{tRNA}}_{{\mathrm{CUG}}}^{{\mathrm{Gln}}}\end{equation*}\end{document} species. These three tRNA species have two structural elements in common, the augmented D-helix and a deletion of nucleotide 47. It appears that the discriminating or nondiscriminating natures of different GluRS enzymes have been derived by the coevolution of protein and tRNA structure. The coexistence of the two GluRS enzymes in one organism may lay the groundwork for the acquisition of the canonical glutaminyl-tRNA synthetase by lateral gene transfer from eukaryotes.

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.

Evolutionary divergence of the archaeal aspartyl-tRNA synthetases into discriminating and nondiscriminating forms

Asparaginyl-tRNA (Asn-tRNA) is generated in nature via two alternate routes, either direct acylation of tRNA with asparagine by asparaginyl-tRNA synthetase (AsnRS) or in a two-step pathway that requires misacylated Asp-tRNAAsn as an intermediate. This misacylated aminoacyl-tRNA is formed by a nondiscriminating aspartyl-tRNA synthetase (AspRS), an enzyme that in addition to forming Asp-tRNAAsp also misacylates tRNAAsn. In contrast, a discriminating AspRS cannot acylate tRNAAsn. It has been suggested that the archaeal AspRS enzymes are nondiscriminating, whereas the bacterial ones discriminate. The archaeal and bacterial AspRS proteins are indeed distinct in sequence and structure. However, we show that both discriminating and nondiscriminating forms of AspRS exist among the archaea. Using unfractionated methanobacterial and pyrococcal tRNA, theMethanothermobacter thermautotrophicus AspRS acylated approximately twice as much tRNA as did AspRS from Pyrococcus kodakaraensis or Ferroplasma acidarmanus. Proof that Asp-tRNAAsn was generated by the methanogen synthetase was the conversion of Asp-tRNA formed by M. thermautotrophicusAspRS to Asn-tRNA by M. thermautotrophicusAsp-tRNAAsn amidotransferase. In contrast, Asp-tRNA formed by the Pyrococcus or Ferroplasma enzymes was not a substrate for the amidotransferase. Also, although all three AspRS enzymes charged tRNAAsp transcripts, only M. thermautotrophicus AspRS aspartylated the tRNAAsntranscript. Genomic analysis provides a rationale for the nature of these enzymes. The mischarging AspRS correlates with the absence in the genome of AsnRS and the presence of Asp-tRNAAsnamidotransferase, employed by the transamidation pathway. In contrast, the discriminating AspRS correlates with the absence of the amidotransferase and the presence of AsnRS, forming Asn-tRNA by direct aminoacylation. The high sequence identity, up to 60% between discriminating and nondiscriminating archaeal AspRSs, suggests that few mutational steps may be necessary to convert the tRNA-discriminating ability of a tRNA synthetase.

Aminoacyl-tRNA synthesis by pre-translational amino acid modification.

Aminoacyl-tRNAs (aa-tRNAs) are essential substrates for ribosomal translation, and aregenerally synthesized by aminoacyl-tRNA synthetases (aaRSs). It was expected earlier thatevery organism would contain a complete set of twenty aaRSs, one for each canonical aminoacid. However, analysis of the many known genome sequences and biochemical studiesrevealed that most organisms lack asparaginyl- and glutaminyl-tRNA synthetases, and thus areunable to attach asparagine and glutamine directly onto their corresponding tRNA. Instead, apre-translational amino acid modification is required to convert Asp-tRNAAsn and Glu-tRNAGlnto the correctly charged Asn-tRNAAsn and Gln-tRNAGln, respectively. This transamidationpathway of amide aa-tRNA synthesis is common in most bacteria and archaea. Unexpectedresults from biochemical, genetic and genomic studies showed that a large variety of differentbacteria rely on tRNA-dependent transamidation for the formation of the amino acidasparagine. Pre-translational modifications are not restricted to asparagine and glutamine butare also found in the biosynthesis of some other aa-tRNAs, such as the initiator tRNA fmettRNAMetiand Sec-tRNASec specifying selenocysteine, the 21st co-translationally inserted aminoacid. tRNA-dependent amino acid modification is also involved in the generation ofaminolevulinic acid, the first precursor for porphyrin biosynthesis in many organisms.

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.

Aminoacyl-tRNA formation in the extreme thermophile Thermus thermophilus.

Abstract. Thermophilic organisms must be capable of accurate translation at temperatures in which the individual components of the translation machinery and also specific amino acids are particularly sensitive. Thermus thermophilus is a good model organism for studies of thermophilic translation because many of the components in this process have undergone structural and biochemical characterization. We have focused on the pathways of aminoacyl-tRNA synthesis for glutamine, asparagine, proline, and cysteine. We show that the T. thermophilus prolyl-tRNA synthetase (ProRS) exhibits cysteinyl-tRNA synthetase (CysRS) activity although the organism also encodes a canonical CysRS. The ProRS requires tRNA for cysteine activation, as is known for the characterized archaeal prolyl-cysteinyl-tRNA synthetase (ProCysRS) enzymes. The heterotrimeric T. thermophilus aspartyl-tRNAAsn amidotransferase can form Gln-tRNA in addition to Asn-tRNA; however, a 13-amino-acid C-terminal truncation of the holoenzyme A subunit is deficient in both activities when assayed with homologous substrates. A survey of codon usage in completed prokaryotic genomes identified a higher Glu:Gln ratio in proteins of thermophiles compared to mesophiles.