Michael J. Hohn

A new phylum of Archaea represented by a nanosized hyperthermophilic symbiont

According to small subunit ribosomal RNA (ss rRNA) sequence comparisons all known Archaea belong to the phyla Crenarchaeota, Euryarchaeota, and—indicated only by environmental DNA sequences—to the ‘Korarchaeota’. Here we report the cultivation of a new nanosized hyperthermophilic archaeon from a submarine hot vent. This archaeon cannot be attached to one of these groups and therefore must represent an unknown phylum which we name ‘Nanoarchaeota’ and species, which we name ‘Nanoarchaeum equitans’. Cells of ‘N. equitans’ are spherical, and only about 400 nm in diameter. They grow attached to the surface of a specific archaeal host, a new member of the genus Ignicoccus. The distribution of the ‘Nanoarchaeota’ is so far unknown. Owing to their unusual ss rRNA sequence, members remained undetectable by commonly used ecological studies based on the polymerase chain reaction. ‘N. equitans’ harbours the smallest archaeal genome; it is only 0.5 megabases in size. This organism will provide insight into the evolution of thermophily, of tiny genomes and of interspecies communication.

Expanding the Genetic Code of Escherichia coli with Phosphoserine

Engineered bacterial translation can be used to direct site-specific insertion of an amino acid into proteins. O-Phosphoserine (Sep), the most abundant phosphoamino acid in the eukaryotic phosphoproteome, is not encoded in the genetic code, but synthesized posttranslationally. Here, we present an engineered system for specific cotranslational Sep incorporation (directed by UAG) into any desired position in a protein by an Escherichia coli strain that harbors a Sep-accepting transfer RNA (tRNASep), its cognate Sep–tRNA synthetase (SepRS), and an engineered EF-Tu (EF-Sep). Expanding the genetic code rested on reengineering EF-Tu to relax its quality-control function and permit Sep-tRNASep binding. To test our system, we synthesized the activated form of human mitogen-activated ERK activating kinase 1 (MEK1) with either one or two Sep residues cotranslationally inserted in their canonical positions (Sep218, Sep222). This system has general utility in protein engineering, molecular biology, and disease research.

Nanoarchaeum equitans creates functional tRNAs from separate genes for their 5′- and 3′-halves

Analysis of the genome sequence of the small hyperthermophilic archaeal parasite Nanoarchaeum equitans has not revealed genes encoding the glutamate, histidine, tryptophan and initiator methionine transfer RNA species. Here we develop a computational approach to genome analysis that searches for widely separated genes encoding tRNA halves that, on the basis of structural prediction, could form intact tRNA molecules. A search of the N. equitans genome reveals nine genes that encode tRNA halves; together they account for the missing tRNA genes. The tRNA sequences are split after the anticodon-adjacent position 37, the normal location of tRNA introns. The terminal sequences can be accommodated in an intervening sequence that includes a 12–14-nucleotide GC-rich RNA duplex between the end of the 5′ tRNA half and the beginning of the 3′ tRNA half. Reverse transcriptase polymerase chain reaction and aminoacylation experiments of N. equitans tRNA demonstrated maturation to full-size tRNA and acceptor activity of the tRNAHis and tRNAGlu species predicted in silico. As the joining mechanism possibly involves tRNA trans-splicing, the presence of an intron might have been required for early tRNA synthesis.

RNA-dependent conversion of phosphoserine forms selenocysteine in eukaryotes and archaea

The trace element selenium is found in proteins as selenocysteine (Sec), the 21st amino acid to participate in ribosome-mediated translation. The substrate for ribosomal protein synthesis is selenocysteinyl-tRNASec. Its biosynthesis from seryl-tRNASec has been established for bacteria, but the mechanism of conversion from Ser-tRNASec remained unresolved for archaea and eukarya. Here, we provide evidence for a different route present in these domains of life that requires the tRNASec-dependent conversion of O-phosphoserine (Sep) to Sec. In this two-step pathway, O-phosphoseryl-tRNASec kinase (PSTK) converts Ser-tRNASec to Sep-tRNASec. This misacylated tRNA is the obligatory precursor for a Sep-tRNA:Sec-tRNA synthase (SepSecS); this protein was previously annotated as SLA/LP. The human and archaeal SepSecS genes complement in vivo an Escherichia coli Sec synthase (SelA) deletion strain. Furthermore, purified recombinant SepSecS converts Sep-tRNASec into Sec-tRNASec in vitro in the presence of sodium selenite and purified recombinant E. coli selenophosphate synthetase (SelD). Phylogenetic arguments suggest that Sec decoding was present in the last universal common ancestor. SepSecS and PSTK coevolved with the archaeal and eukaryotic lineages, but the history of PSTK is marked by several horizontal gene transfer events, including transfer to non-Sec-decoding Cyanobacteria and fungi.

From one amino acid to another: tRNA-dependent amino acid biosynthesis

Aminoacyl-tRNAs (aa-tRNAs) are the essential substrates for translation. Most aa-tRNAs are formed by direct aminoacylation of tRNA catalyzed by aminoacyl-tRNA synthetases. However, a smaller number of aa-tRNAs (Asn-tRNA, Gln-tRNA, Cys-tRNA and Sec-tRNA) are made by synthesizing the amino acid on the tRNA by first attaching a non-cognate amino acid to the tRNA, which is then converted to the cognate one catalyzed by tRNA-dependent modifying enzymes. Asn-tRNA or Gln-tRNA formation in most prokaryotes requires amidation of Asp-tRNA or Glu-tRNA by amidotransferases that couple an amidase or an asparaginase to liberate ammonia with a tRNA-dependent kinase. Both archaeal and eukaryotic Sec-tRNA biosynthesis and Cys-tRNA synthesis in methanogens require O-phosophoseryl-tRNA formation. For tRNA-dependent Cys biosynthesis, O-phosphoseryl-tRNA synthetase directly attaches the amino acid to the tRNA which is then converted to Cys by Sep-tRNA: Cys-tRNA synthase. In Sec-tRNA synthesis, O-phosphoseryl-tRNA kinase phosphorylates Ser-tRNA to form the intermediate which is then modified to Sec-tRNA by Sep-tRNA:Sec-tRNA synthase. Complex formation between enzymes in the same pathway may protect the fidelity of protein synthesis. How these tRNA-dependent amino acid biosynthetic routes are integrated into overall metabolism may explain why they are still retained in so many organisms.

Rewiring translation for elongation factor Tu-dependent selenocysteine incorporation

Selenium is an essential micronutrient for animals.[1] Humans contain 25 presumably essential selenoproteins[2] in which selenium is found in the form of Sec.[3] In this 21st genetically encoded amino acid[4] the thiol moiety of Cys is replaced by a selenol group. In all Sec-decoding organisms, Sec biosynthesis (Scheme 1B) starts with the acylation of tRNASec by seryl-tRNA synthetase (SerRS) to form Ser-tRNASec (reviewed in[5]). In bacteria, conversion of Ser-tRNASec to Sec-tRNASec is achieved by SelA (reviewed in[4]). In contrast, archaea and eukaryotes employ an additional phosphorylation step. O-phosphoseryl-tRNASec kinase (PSTK) phosphorylates the tRNA-bound Ser moiety of Ser-tRNASec to form O-phosphoseryl-tRNASec (Sep-tRNASec),[6] the substrate for SepSecS that catalyzes the tRNA-dependent Sep to Sec conversion.[7] The selenium donor for both SelA and SepSecS is selenophosphate (reviewed in[4, 7b]).

Toward understanding phosphoseryl-tRNACys formation: The crystal structure of Methanococcus maripaludis phosphoseryl-tRNA synthetase

A number of archaeal organisms generate Cys-tRNACys in a two-step pathway, first charging phosphoserine (Sep) onto tRNACys and subsequently converting it to Cys-tRNACys. We have determined, at 3.2-Å resolution, the structure of the Methanococcus maripaludis phosphoseryl-tRNA synthetase (SepRS), which catalyzes the first step of this pathway. The structure shows that SepRS is a class II, α4 synthetase whose quaternary structure arrangement of subunits closely resembles that of the heterotetrameric (αβ)2 phenylalanyl-tRNA synthetase (PheRS). Homology modeling of a tRNA complex indicates that, in contrast to PheRS, a single monomer in the SepRS tetramer may recognize both the acceptor terminus and anticodon of a tRNA substrate. Using a complex with tungstate as a marker for the position of the phosphate moiety of Sep, we suggest that SepRS and PheRS bind their respective amino acid substrates in dissimilar orientations by using different residues.

How an obscure archaeal gene inspired the discovery of selenocysteine biosynthesis in humans

Selenocysteine (Sec) is the 21st genetically encoded amino acid found in organisms from all three domains of life. Sec biosynthesis is unique in that it always proceeds from an aminoacyl‐tRNA precursor. Even though Sec biosynthesis in bacteria was established almost two decades ago, only recently the pathway was elucidated in archaea and eukaryotes. While other aspects of Sec biology have been reviewed previously (Allmang and Krol, Biochimie 2006;88:1561–1571, Hatfield et al., Prog Nucleic Acid Res Mol Biol 2006;81:97–142, Squires and Berry, IUBMB Life 2008;60:232–235), here we review the biochemistry and evolution of Sec biosynthesis and coding and show how the knowledge of an archaeal cysteine biosynthesis pathway helped to uncover the route to Sec formation in archaea and eukaryotes.

A tRNA-dependent cysteine biosynthesis enzyme recognizes the selenocysteine-specific tRNA in Escherichia coli

The essential methanogen enzyme Sep‐tRNA:Cys‐tRNA synthase (SepCysS) converts O‐phosphoseryl‐tRNACys (Sep‐tRNACys) into Cys‐tRNACys in the presence of a sulfur donor. Likewise, Sep‐tRNA:Sec‐tRNA synthase converts O‐phosphoseryl‐tRNASec (Sep‐tRNASec) to selenocysteinyl‐tRNASec (Sec‐tRNASec) using a selenium donor. While the Sep moiety of the aminoacyl‐tRNA substrates is the same in both reactions, tRNACys and tRNASec differ greatly in sequence and structure. In an Escherichia coli genetic approach that tests for formate dehydrogenase activity in the absence of selenium donor we show that Sep‐tRNASec is a substrate for SepCysS. Since Sec and Cys are the only active site amino acids known to sustain FDH activity, we conclude that SepCysS converts Sep‐tRNASec to Cys‐tRNASec, and that Sep is crucial for SepCysS recognition.

Genetic analysis of selenocysteine biosynthesis in the archaeon Methanococcus maripaludis.

In Archaea selenocysteine (Sec) is synthesized in three steps. First seryl‐tRNA synthetase acylates tRNASec with serine to generate Ser‐tRNASec. Then phosphoseryl‐tRNASec kinase (PSTK) forms Sep‐tRNASec, which is converted to Sec‐tRNASec by Sep‐tRNA:Sec‐tRNA synthase (SepSecS) in the presence of selenophosphate produced by selenophosphate synthetase (SelD). A complete in vivo analysis of the archaeal Sec biosynthesis pathway is still unavailable, and the existence of a redundant pathway or of a rescue mechanism based on the conversion of Sep‐tRNASec to Cys‐tRNASec during selenium starvation, cannot be excluded. Here we present a mutational analysis of Sec biosynthesis in Methanococcus maripaludis strain Mm900. Sec formation is abolished upon individually deleting the genes encoding SelD, PSTK or SepSecS; the resulting mutant strains could no longer grow on formate while growth with H2 + CO2 remained unaffected. However, deletion of the PSTK and SepSecS genes was not possible unless the selenium‐free [NiFe]‐hydrogenases Frc and Vhc were expressed. This required the prior deletion of either the gene encoding SelD or that of HrsM, a LysR‐type regulator suppressing transcription of the frc and vhc operons in the presence of selenium. These results show that M. maripaludis Mm900 is facultatively selenium‐dependent with a single pathway of Sec‐tRNASec formation.