Archaeal tRNA-Splicing Endonuclease as an Effector for RNA Recombination and Novel Trans-Splicing Pathways in Eukaryotes

We have characterized a homodimeric tRNA endonuclease from the euryarchaeota Ferroplasma acidarmanus (FERAC), a facultative anaerobe which can grow at temperatures ranging from 35 to 42 °C. This enzyme, contrary to the eukaryal tRNA endonucleases and the homotetrameric Methanocaldococcus jannaschii (METJA) homologs, is able to cleave minimal BHB (bulge–helix–bulge) substrates at 30 °C. The expression of this enzyme in Schizosaccharomyces pombe (SCHPO) enables the use of its properties as effectors by inserting BHB motif introns into hairpin loops normally seen in mRNA transcripts. In addition, the FERAC endonuclease can create proteins with new functionalities through the recombination of protein domains.

The Oxoglutarate Binding Site and Regulatory Mechanism Are Conserved in Ammonium Transporter Inhibitors GlnKs from Methanococcales

Protein inhibition is a natural regulatory process to control cellular metabolic fluxes. PII-family signal-transducing effectors are in this matter key regulators of the nitrogen metabolism. Their interaction with their various targets is governed by the cellular nitrogen level and the energy charge. Structural studies on GlnK, a PII-family inhibitor of the ammonium transporters (Amt), showed that the T-loops responsible for channel obstruction are displaced upon the binding of 2-oxoglutarate, magnesium and ATP in a conserved cleft. However, GlnK from Methanocaldococcus jannaschii was shown to bind 2-oxoglutarate on the tip of its T-loop, causing a moderate disruption to GlnK–Amt interaction, raising the question if methanogenic archaea use a singular adaptive strategy. Here we show that membrane fractions of Methanothermococcus thermolithotrophicus released GlnKs only in the presence of Mg-ATP and 2-oxoglutarate. This observation led us to structurally characterize the two GlnK isoforms apo or in complex with ligands. Together, our results show that the 2-oxoglutarate binding interface is conserved in GlnKs from Methanococcales, including Methanocaldococcus jannaschii, emphasizing the importance of a free carboxy-terminal group to facilitate ligand binding and to provoke the shift of the T-loop positions.

Filamentous Chaperone Protein-Based Hydrogel Stabilizes Enzymes against Thermal Inactivation

We report a filamentous chaperone-based protein hydrogel capable of stabilizing enzymes against thermal inactivation. The hydrogel backbone consists of a thermostable chaperone protein, the gamma-prefoldin (γPFD) from Methanocaldococcus jannaschii, which...

Growth Kinetics, Carbon Isotope Fractionation, and Gene Expression in the HyperthermophileMethanocaldococcus jannaschiiduring Hydrogen-Limited Growth and Interspecies Hydrogen Transfer

ABSTRACTHyperthermophilic methanogens are often H2limited in hot subseafloor environments, and their survival may be due in part to physiological adaptations to low H2conditions and interspecies H2transfer. The hyperthermophilic methanogenMethanocaldococcus jannaschiiwas grown in monoculture at high (80 to 83 μM) and low (15 to 27 μM) aqueous H2concentrations and in coculture with the hyperthermophilic H2producerThermococcus paralvinellae. The purpose was to measure changes in growth and CH4production kinetics, CH4fractionation, and gene expression inM. jannaschiiwith changes in H2flux. Growth and cell-specific CH4production rates ofM. jannaschiidecreased with decreasing H2availability and decreased further in coculture. However, cell yield (cells produced per mole of CH4produced) increased 6-fold whenM. jannaschiiwas grown in coculture rather than monoculture. Relative to high H2concentrations, isotopic fractionation of CO2to CH4(εCO2-CH4) was 16‰ larger for cultures grown at low H2concentrations and 45‰ and 56‰ larger forM. jannaschiigrowth in coculture on maltose and formate, respectively. Gene expression analyses showed H2-dependent methylene-tetrahydromethanopterin (H4MPT) dehydrogenase expression decreased and coenzyme F420-dependent methylene-H4MPT dehydrogenase expression increased with decreasing H2availability and in coculture growth. In coculture, gene expression decreased for membrane-bound ATP synthase and hydrogenase. The results suggest that H2availability significantly affects the CH4and biomass production and CH4fractionation by hyperthermophilic methanogens in their native habitats.IMPORTANCEHyperthermophilic methanogens and H2-producing heterotrophs are collocated in high-temperature subseafloor environments, such as petroleum reservoirs, mid-ocean ridge flanks, and hydrothermal vents. Abiotic flux of H2can be very low in these environments, and there is a gap in our knowledge about the origin of CH4in these habitats. In the hyperthermophileMethanocaldococcus jannaschii, growth yields increased as H2flux, growth rates, and CH4production rates decreased. The same trend was observed increasingly with interspecies H2transfer betweenM. jannaschiiand the hyperthermophilic H2producerThermococcus paralvinellae. With decreasing H2availability, isotopic fractionation of carbon during methanogenesis increased, resulting in isotopically more negative CH4with a concomitant decrease in H2-dependent methylene-tetrahydromethanopterin dehydrogenase gene expression and increase in F420-dependent methylene-tetrahydromethanopterin dehydrogenase gene expression. The significance of our research is in understanding the nature of hyperthermophilic interspecies H2transfer and identifying biogeochemical and molecular markers for assessing the physiological state of methanogens and possible source of CH4in natural environments.

tRNA Modification Profiles and Codon-Decoding Strategies in Methanocaldococcus jannaschii

ABSTRACT tRNAs play a critical role in mRNA decoding, and posttranscriptional modifications within tRNAs drive decoding efficiency and accuracy. The types and positions of tRNA modifications in model bacteria have been extensively studied, and tRNA modifications in a few eukaryotic organisms have also been characterized and localized to particular tRNA sequences. However, far less is known regarding tRNA modifications in archaea. While the identities of modifications have been determined for multiple archaeal organisms, Haloferax volcanii is the only organism for which modifications have been extensively localized to specific tRNA sequences. To improve our understanding of archaeal tRNA modification patterns and codon-decoding strategies, we have used liquid chromatography and tandem mass spectrometry to characterize and then map posttranscriptional modifications on 34 of the 35 unique tRNA sequences of Methanocaldococcus jannaschii. A new posttranscriptionally modified nucleoside, 5-cyanomethyl-2-thiouridine (cnm5s2U), was discovered and localized to position 34. Moreover, data consistent with wyosine pathway modifications were obtained beyond the canonical tRNAPhe as is typical for eukaryotes. The high-quality mapping of tRNA anticodon loops enriches our understanding of archaeal tRNA modification profiles and decoding strategies. IMPORTANCE While many posttranscriptional modifications in M. jannaschii tRNAs are also found in bacteria and eukaryotes, several that are unique to archaea were identified. By RNA modification mapping, the modification profiles of M. jannaschii tRNA anticodon loops were characterized, allowing a comparative analysis with H. volcanii modification profiles as well as a general comparison with bacterial and eukaryotic decoding strategies. This general comparison reveals that M. jannaschii, like H. volcanii, follows codon-decoding strategies similar to those used by bacteria, although position 37 appears to be modified to a greater extent than seen in H. volcanii.

Dissecting the Contribution of Release Factor Interactions to Amber Stop Codon Reassignment Efficiencies of the Methanocaldococcus jannaschii Orthogonal Pair

Non-canonical amino acids (ncAAs) are finding increasing use in basic biochemical studies and biomedical applications. The efficiency of ncAA incorporation is highly variable, as a result of competing system composition and codon context effects. The relative quantitative contribution of the multiple factors affecting incorporation efficiency are largely unknown. This manuscript describes the use of green fluorescent protein (GFP) reporters to quantify the efficiency of amber codon reassignment using the Methanocaldococcus jannaschii orthogonal pair system, commonly employed for ncAA incorporation, and quantify the contribution of release factor 1 (RF1) to the overall efficiency of amino acid incorporation. The efficiencies of amber codon reassignments were quantified at eight positions in GFP and evaluated in multiple combinations. The quantitative contribution of RF1 competition to reassignment efficiency was evaluated through comparisons of amber codon suppression efficiencies in normal and genomically recoded Escherichia coli strains. Measured amber stop codon reassignment efficiencies for eight single stop codon GFP variants ranged from 51 to 117% in E. coli DH10B and 76 to 104% in the RF1 deleted E. coli C321.ΔA.exp. Evaluation of efficiency changes in specific sequence contexts in the presence and absence of RF1 suggested that RF1 specifically interacts with +4 Cs and that the RF1 interactions contributed approximately half of the observed sequence context-dependent variation in measured reassignment efficiency. Evaluation of multisite suppression efficiencies suggests that increasing demand for translation system components limits multisite incorporation in cells with competing RF1.