In vitro biosynthesis of a universal t6A tRNA modification in Archaea and Eukarya

N⁶-threonylcarbamoyladenosine (t⁶A) is a modified nucleotide found in all transfer RNAs (tRNAs) decoding codons starting with adenosine. Its role is to facilitate codon–anticodon pairing and to prevent frameshifting during protein synthesis. Genetic studies demonstrated that two universal proteins, Kae1/YgjD and Sua5/YrdC, are necessary for t⁶A synthesis in Saccharomyces cerevisiae and Escherichia coli. In Archaea and Eukarya, Kae1 is part of a conserved protein complex named kinase, endopeptidase and other proteins of small size (KEOPS), together with three proteins that have no bacterial homologues. Here, we reconstituted for the first time an in vitro system for t⁶A modification in Archaea and Eukarya, using purified KEOPS and Sua5. We demonstrated binding of tRNAs to archaeal KEOPS and detected two distinct adenosine triphosphate (ATP)-dependent steps occurring in the course of the synthesis. Our data, together with recent reconstitution of an in vitro bacterial system, indicated that t⁶A cannot be catalysed by Sua5/YrdC and Kae1/YgjD alone but requires accessory proteins that are not universal. Remarkably, we observed interdomain complementation when bacterial, archaeal and eukaryotic proteins were combined in vitro, suggesting a conserved catalytic mechanism for the biosynthesis of t⁶A in nature. These findings shed light on the reaction mechanism of t⁶A synthesis and evolution of molecular systems that promote translation fidelity in present-day cells.     

The ATP-mediated formation of the YgjD–YeaZ–YjeE complex is required for the biosynthesis of tRNA t6A in Escherichia coli

The essential and universal N⁶-threonylcarbamoyladenosine (t⁶A) modification at position 37 of ANN-decoding tRNAs plays a pivotal role in translational fidelity through enhancement of the cognate codon recognition and stabilization of the codon–anticodon interaction. In Escherichia coli, the YgjD (TsaD), YeaZ (TsaB), YjeE (TsaE) and YrdC (TsaC) proteins are necessary and sufficient for the in vitro biosynthesis of t⁶A, using tRNA, ATP, L-threonine and bicarbonate as substrates. YrdC synthesizes the short-lived L-threonylcarbamoyladenylate (TCA), and YgjD, YeaZ and YjeE cooperate to transfer the L-threonylcarbamoyl-moiety from TCA onto adenosine at position 37 of substrate tRNA. We determined the crystal structure of the heterodimer YgjD–YeaZ at 2.3 Å, revealing the presence of an unexpected molecule of ADP bound at an atypical site situated at the YgjD–YeaZ interface. We further showed that the ATPase activity of YjeE is strongly activated by the YgjD–YeaZ heterodimer. We established by binding experiments and SAXS data analysis that YgjD–YeaZ and YjeE form a compact ternary complex only in presence of ATP. The formation of the ternary YgjD–YeaZ–YjeE complex is required for the in vitro biosynthesis of t⁶A but not its ATPase activity.     

Cross Kingdom Functional Conservation of the Core Universally Conserved Threonylcarbamoyladenosine tRNA Synthesis Enzymes

Threonylcarbamoyladenosine (t⁶A) is a universal modification located in the anticodon stem-loop of tRNAs. In yeast, both cytoplasmic and mitochondrial tRNAs are modified. The cytoplasmic t⁶A synthesis pathway was elucidated and requires Sua5p, Kae1p, and four other KEOPS complex proteins. Recent in vitro work suggested that the mitochondrial t⁶A machinery of Saccharomyces cerevisiae is composed of only two proteins, Sua5p and Qri7p, a member of the Kae1p/TsaD family (L. C. K. Wan et al., Nucleic Acids Res. 41:6332–6346, 2013, http://dx.doi.org/10.1093/nar/gkt322). Sua5p catalyzes the first step leading to the threonyl-carbamoyl-AMP intermediate (TC-AMP), while Qri7 transfers the threonyl-carbamoyl moiety from TC-AMP to tRNA to form t⁶A. Qri7p localizes to the mitochondria, but Sua5p was reported to be cytoplasmic. We show that Sua5p is targeted to both the cytoplasm and the mitochondria through the use of alternative start sites. The import of Sua5p into the mitochondria is required for this organelle to be functional, since the TC-AMP intermediate produced by Sua5p in the cytoplasm is not transported into the mitochondria in sufficient amounts. This minimal t⁶A pathway was characterized in vitro and, for the first time, in vivo by heterologous complementation studies in Escherichia coli. The data revealed a potential for TC-AMP channeling in the t⁶A pathway, as the coexpression of Qri7p and Sua5p is required to complement the essentiality of the E. coli tsaD mutant. Our results firmly established that Qri7p and Sua5p constitute the mitochondrial pathway for the biosynthesis of t⁶A and bring additional advancement in our understanding of the reaction mechanism.     

Yeast KEOPS complex regulates telomere length independently of its t6A modification function

In Saccharomyces cerevisiae, the highly conserved Sua5 and KEOPS complex(including five subunits Kae1,Bud32, Cgi121, Pcc1 and Gon7) catalyze a universal t RNA modification, namely N~6-threonylcarbamoyladenosine(t~6A), and regulate telomere replication and recombination. However, whether telomere regulation function of Sua5 and KEOPS complex depends on the t~6A modification activity remains unclear. Here we show that Sua5 and KEOPS regulate telomere length in the same genetic pathway.Interestingly, the telomere length regulation by KEOPS is independent of its t~6A biosynthesis activity.Cytoplasmic overexpression of Qri7, a functional counterpart of KEOPS in mitochondria, restores cytosolic t RNA t~6A modification and cell growth, but is not sufficient to rescue telomere length in the KEOPS mutant kae1△ cells, indicating that a t~6A modification-independent function is responsible for the telomere regulation. The results of our in vitro biochemical and in vivo genetic assays suggest that telomerase RNA TLC1 might not be modified by Sua5 and KEOPS. Moreover, deletion of KEOPS subunits results in a dramatic reduction of telomeric G-overhang, suggesting that KEOPS regulates telomere length by promoting G-overhang generation. These findings support a model in which KEOPS regulates telomere replication independently of its function on t RNA modification.     

Ribosomal protein modification in Escherichia coli. I. A mutant lacking the N-terminal acetylation of protein S5 exhibits thermosensitivity.

A thermosensitive mutant (JE386) of Escherichia coli which harbours an alteration in protein S5 of the smaller ribosomal subunit has been isolated. Genetic studies have shown that the lesion causing thermosensitivity also causes the alteration in protein S5, and that this mutation is not in the structural gene for S5 (rpsE). Hence the mutation has been termed rimJ (ribosomal modification). Protein-chemical studies of protein S5 purified from JE386 and its wild-type parent indicated an alteration in the N-terminal tryptic peptide. Amino acid sequence analysis of the N-terminal peptides showed complete homology between wild-type and mutant, suggesting that the N-terminal modification (acetylation) of the parent was absent in the mutant. Gradient transmission mapping has located the rimJ mutation at 31 minutes on the current E. coli genetic map. By constructing a derivative of the mutant heterozygous for rimJ, it has been found that the wild-type allele is dominant over the mutant one. Ts⁺ revertants of JE386 have been isolated which show either a wild-type ribosomal protein electrophoresis pattern, or an additional alteration in either protein S4 or S5. The mutations in S4 and S5 may compensate the lesion caused by the rimJ mutation of JE386, that is even though the N-terminus of S5 remains unacetylated, bacteria can grow at 42 °C. Furthermore, a mutation near or at strA carried by JE386 has been found to be involved in the phenotypic expression of the rimJ mutation. This mutation was also found to be present in four other strA mutants. Possible implications of the modification of ribosomal proteins in vivo are discussed.     

Bioaccumulation of heavy metals in Escherichia coli expressing an inducible synthetic human metallothionein gene

A synthetic DNA coding for human hepatic metallothionein (HMT), fraction MT-2, has been cloned in the plasmid vector pING 1 and expressed in E. coli. Upon induction with arabinose, an araB′-HMT fusion protein is synthesized. The fusion protein is produced to a level of approximately 8% of the total protein in E. coli, with an apparent half-life of 50 min. Without arabinose, no fusion protein is expressed, demonstrating that the expression system is tightly regulated. In the presence of ¹⁰⁹Cd, the fusion protein binds the metal ion in vitro. Concurrently, a direct correlation is found between the expression in E. coli of the araB′-HMT fusion protein which binds Cd²⁺ in vitro and the bioaccumulation of Cd²⁺ in vivo. The bioaccumulation of Cu⁺ in E. coli, when the fusion protein is produced, has also been established.     

Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification

N⁶-threonylcarbamoyladenosine (t⁶A) is a universal tRNA modification essential for normal cell growth and accurate translation. In Archaea and Eukarya, the universal protein Sua5 and the conserved KEOPS/EKC complex together catalyze t⁶A biosynthesis. The KEOPS/EKC complex is composed of Kae1, a universal metalloprotein belonging to the ASHKA superfamily of ATPases; Bud32, an atypical protein kinase and two small proteins, Cgi121 and Pcc1. In this study, we investigated the requirement and functional role of KEOPS/EKC subunits for biosynthesis of t⁶A. We demonstrated that Pcc1, Kae1 and Bud32 form a minimal functional unit, whereas Cgi121 acts as an allosteric regulator. We confirmed that Pcc1 promotes dimerization of the KEOPS/EKC complex and uncovered that together with Kae1, it forms the tRNA binding core of the complex. Kae1 binds l-threonyl-carbamoyl-AMP intermediate in a metal-dependent fashion and transfers the l-threonyl-carbamoyl moiety to substrate tRNA. Surprisingly, we found that Bud32 is regulated by Kae1 and does not function as a protein kinase but as a P-loop ATPase possibly involved in tRNA dissociation. Overall, our data support a mechanistic model in which the final step in the biosynthesis of t⁶A relies on a strictly catalytic component, Kae1, and three partner proteins necessary for dimerization, tRNA binding and regulation.     

Commonality and diversity in tRNA substrate recognition in t6A biogenesis by eukaryotic KEOPSs

N ⁶-Threonylcarbamoyladenosine (t⁶A) is a universal and pivotal tRNA modification. KEOPS in eukaryotes participates in its biogenesis, whose mutations are connected with Galloway-Mowat syndrome. However, the tRNA substrate selection mechanism by KEOPS and t⁶A modification function in mammalian cells remain unclear. Here, we confirmed that all ANN-decoding human cytoplasmic tRNAs harbor a t⁶A moiety. Using t⁶A modification systems from various eukaryotes, we proposed the possible coevolution of position 33 of initiator tRNA^Met and modification enzymes. The role of the universal CCA end in t⁶A biogenesis varied among species. However, all KEOPSs critically depended on C32 and two base pairs in the D-stem. Knockdown of the catalytic subunit OSGEP in HEK293T cells had no effect on the steady-state abundance of cytoplasmic tRNAs but selectively inhibited tRNA^Ile aminoacylation. Combined with in vitro aminoacylation assays, we revealed that t⁶A functions as a tRNA^Ile isoacceptor-specific positive determinant for human cytoplasmic isoleucyl-tRNA synthetase (IARS1). t⁶A deficiency had divergent effects on decoding efficiency at ANN codons and promoted +1 frameshifting. Altogether, our results shed light on the tRNA recognition mechanism, revealing both commonality and diversity in substrate recognition by eukaryotic KEOPSs, and elucidated the critical role of t⁶A in tRNA^Ile aminoacylation and codon decoding in human cells.     

The PecM protein is necessary for the DNA-binding capacity of the PecS repressor, one of the regulators of virulence-factor synthesis in Erwinia chrysanthemi

The pecS regulatory locus is responsible for the down-expression of many virulence genes in Erwinia chrysanthemi. This locus consists of two genes, pecS and pecM, divergently transcribed. Genetic evidence indicates that the PecM protein modulates the regulatory activity of PecS. Purification and characterization of PecS, expressed either from E. coli, from the wild-type E. chrysanthemi strain or from a pecM mutant, showed that the PecS protein produced in these three genetic backgrounds displays the same biochemical properties. Band-shift assay analysis with the three PecS isoforms confirmed the involvement of the PecM protein in modulating the PecS DNA-binding capacity. Moreover, determination of the Kd_app for operator regions of the PecS protein, produced either by the wild-type E. chrysanthemi or by E. coli, reveals similar affinities. Thus, in E. coli, there is likely to be at least one other PecM-like protein able to cross-react with the E. chrysanthemi PecS protein.     

Crystal structure and functional implications of the tandem-type universal stress protein UspE from <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background

The universal stress proteins (USP) family member UspE is a tandem-type USP that consists of two Usp domains. The UspE expression levels of the Escherichia coli (E. coli) become elevated in response to oxidative stress and DNA damaging agents, including exposure to mitomycin C, cadmium, and hydrogen peroxide. It has been shown that UspA family members are survival factors during cellular growth arrest. The structures and functions of the UspA family members control the growth of E. coli in animal hosts. While several UspA family members have known structures, the structure of E. coli UspE remains to be elucidated.

Results

To understand the biochemical function of UspE, we have determined the crystal structure of E. coli UspE at 3.2 Å resolution. The asymmetric unit contains two protomers related by a non-crystallographic symmetry, and each protomer contains two tandem Usp domains. The crystal structure shows that UspE is folded into a fan-shaped structure similar to that of the tandem-type Usp protein PMI1202 from Proteus mirabilis, and it has a hydrophobic cavity that binds its ligand. Structural analysis revealed that E. coli UspE has two metal ion binding sites, and isothermal titration calorimetry suggested the presence of two Cd²⁺ binding sites with a K_d value of 38.3–242.7 μM. Structural analysis suggested that E. coli UspE has two Cd²⁺ binding sites (Site I: His117, His 119; Site II: His193, His244).

Conclusion

The results show that the UspE structure has a hydrophobic pocket. This pocket is strongly bound to an unidentified ligand. Combined with a previous study, the ligand is probably related to an intermediate in lipid A biosynthesis. Subsequently, sequence analysis found that UspE has an ATP binding motif (Gly²⁶⁹- X₂-Gly²⁷²-X₉-Gly²⁸²-Asn) in its C-terminal domain, which was confirmed by in vitro ATPase activity monitored using Kinase-Glo® Luminescent Kinase Assay. However, the residues constituting this motif were disordered in the crystal structure, reflecting their intrinsic flexibility. ITC experiments revealed that the UspE probably has two Cd²⁺ binding sites. The His117, His 119, His193, and His244 residues within the β-barrel domain are necessary for Cd²⁺ binding to UspE protein. As mentioned above, USPs are associated with several functions, such as cadmium binding, ATPase function, and involvement in lipid A biosynthesis by some unknown way.

     

The yfiC gene of E. coli encodes an adenine-N6 methyltransferase that specifically modifies A37 of tRNA1Val(cmo5UAC)

Transfer RNA is highly modified. Nucleotide 37 of the anticodon loop is represented by various modified nucleotides. In Escherichia coli, the valine-specific tRNA (cmo⁵UAC) contains a unique modification, N⁶-methyladenosine, at position 37; however, the enzyme responsible for this modification is unknown. Here we demonstrate that the yfiC gene of E. coli encodes an enzyme responsible for the methylation of A37 in tRNA₁^Val. Inactivation of yfiC gene abolishes m⁶A formation in tRNA₁^Val, while expression of the yfiC gene from a plasmid restores the modification. Additionally, unmodified tRNA₁^Val can be methylated by recombinant YfiC protein in vitro. Although the methylation of m⁶A in tRNA₁^Val by YfiC has little influence on the cell growth under standard conditions, the yfiC gene confers a growth advantage under conditions of osmotic and oxidative stress.     

A variable automatic recording device for measuring phytochrome with the Perkin-Elmer spectrophotometer model 356

A method is described for the quantitative analysis and preparative isolation of N-[N-methyl-N-(9-β-ribofuranosylpurin-6-yl)carbamoyl]threonine (mt⁶A), a rare modified nucleoside constituent of transfer RNA. This method is based on the selective retention of mt⁶A and its parent compound, t⁶A, on Dowex-1 at pH 7.8, allowing these two nucleosides to be readily concentrated from the mixture of nucleosides resulting when tRNA is hydrolyzed by a combination of snake venom enzymes and E. coli alkaline phosphatase. The content of mt⁶A in wheat embryo and E. coli tRNA was found to be about 0.025 mole %, which is roughly one-tenth the t⁶A content of the tRNA of these two organisms. Since this indicates that only about 1 in 50 chains can contain a residue of mt⁶A, this nucleoside may be confined to a single isoaccepting species of transfer RNA in both E. coli and wheat embryo. No mt⁶A could be detected in either baker's or brewer's yeast tRNA by the method described. Either mt⁶A is entirely absent from yeast tRNA or it occurs in a form which does not adsorb to Dowex-1 during fractionation of hydrolysates of yeast tRNA. No t⁶A or mt⁶A could be detected in the 18 S + 26 S ribosomal ribonucleates of wheat embryo.     

The Methyl Group of the N6-Methyl-N6-Threonylcarbamoyladenosine in tRNA of Escherichia coli Modestly Improves the Efficiency of the tRNA

tRNA species that read codons starting with adenosine (A) contain N⁶-threonylcarbamoyladenosine (t⁶A) derivatives adjacent to and 3′ of the anticodons from all organisms. In Escherichia coli there are 12 such tRNA species of which two (tRNA_GGU^Thr1 and tRNA_GGU^Thr3) have the t⁶A derivative N⁶-methyl-N⁶-threonylcarbamoyladenosine (m⁶t⁶A37). We have isolated a mutant of E. coli that lacks the m⁶t⁶A37 in these two tRNA_GGU^Thr species. These tRNA species in the mutant are likely to have t⁶A37 instead of m⁶t⁶A37. We show that the methyl group of m⁶t⁶A37 originates from S-adenosyl-l-methionine and that the gene (tsaA) which most likely encodes tRNA(m⁶t⁶A37)methyltransferase is located at min 4.6 on the E. coli chromosomal map. The growth rate of the cell, the polypeptide chain elongation rate, and the selection of Thr-tRNA_GGU^Thr to the ribosomal A site programmed with either of the cognate codons ACC and ACU were the same for the tsaA1 mutant as for the congenic wild-type strain. The expression of the threonine operon is regulated by an attenuator which contains in its leader mRNA seven ACC codons that are read by these two m⁶t⁶A37-containing tRNA_GGU^Thr species. We show that the tsaA1 mutation resulted in a twofold derepression of this operon, suggesting that the lack of the methyl group of m⁶t⁶A37 in tRNA_GGU^Thr slightly reduces the efficiency of this tRNA to read cognate codon ACC.All tRNA species from the three domains, Archaea, Bacteria, and Eucarya, contain modified nucleosides, which are derivatives of the four nucleosides, adenosine, guanosine, cytidine, and uridine. At present, more than 79 different modified nucleosides from the tRNA of various organisms have been characterized (23). Some of these are present in tRNA from only one domain, but a few are present in the same subset of and at the same position in the tRNAs from all three domains (3). One such conserved group of modified nucleosides is the threonylated adenosine (t⁶A) derivatives. These modified adenosines are present adjacent to and 3′ of the anticodon (position 37) in the subset of tRNAs that reads codons starting with A. The universal presence of t⁶A derivatives suggests that these kinds of modifications may have been present in the tRNA of the progenitor, unless a convergent evolution has occurred. This conservation also suggests that the functions of these modified nucleosides may be principally the same in all organisms.In Escherichia coli, the t⁶A37 derivative N⁶-methyl-N⁶- threonylcarbamoyladenosine (m⁶t⁶A37) is present in only two tRNA species, the tRNA_GGU^Thr species, with the same anticodon (20). Threonine is the precursor in the synthesis of t⁶A (10, 32), and in vitro threonylation requires carbonate and ATP (15, 21). Here we show that the methyl group of m⁶t⁶A37 originates from methionine. So far, no mutant deficient in any t⁶A37 derivative has been characterized. As a first step to elucidate the syntheses of these groups of modified nucleosides and their roles in vivo, we have isolated and characterized a mutant deficient in the synthesis of m⁶t⁶A37. We show that the tsaA gene most likely encodes the tRNA(m⁶t⁶A37)methyltransferase that transfers a methyl group from S-adenosylmethionine (AdoMet) to the two tRNA_GGU^Thr species containing the t⁶A moiety. The tsaA gene was localized to the 4.6 min site on the E. coli chromosome. We also show that the methyl group of m⁶t⁶A37 slightly improves the translational efficiency of the two tRNA_GGU^Thr species.