Association of an Aminoacyl-tRNA Synthetase with a Putative Metabolic Protein in Archaea

Lipman, Richard S. A.; Chen, Jing; Evilia, Caryn; Vitseva, Olga; Hou, Ya‐Ming

doi:10.1021/bi0344533

Cited by 23 publications

(48 citation statements)

References 47 publications

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“…Whereas HMD itself or known similar factors were not detected here, ProRS did identify a subunit of a protein that under certain conditions provides reduced coenzyme F420 for methylene-H 4 MPT formation. The independent identification in this study of interactions between ProRS and enzymes contributing to the same step in methanogenesis as HMD supports the proposed link between metabolism and decoding in Archaea (18), although the role of such a connection remains unknown.…”

Section: Discussionsupporting

confidence: 77%

“…Proteins were identified in the ProRS-mediated two-hybrid screens that are parts of the complexes carrying out three of the eight steps. The two-hybrid screen did not identify the protein HMD, which in M. jannaschii co-purified with ProRS (18). HMD catalyzes the conversion of methenyl-H 4 MPT to methylene-H 4 MPT, the fourth step in the reduction of CO 2 to CH 4 in M. thermautotrophicus (33).…”

Section: Discussionmentioning

confidence: 99%

“…Methanocaldococcus jannaschii ProRS was co-purified with the H 2 -forming N 5 -N 10 -methylene tetrahydromethanopterin dehydrogenase (HMD), a component of the methanogenesis pathway (18). ProRS and HMD were also used for co-immunoprecipitation experiments with recombinant proteins, leading to the proposal that HMD specifically interacts not only with ProRS but also with lysyl-tRNA synthetase and aspartyl-tRNA synthetase.…”

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confidence: 99%

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Association between Archaeal Prolyl- and Leucyl-tRNA Synthetases Enhances tRNAPro Aminoacylation

Prætorius-Ibba¹,

Rogers²,

Samson³

et al. 2005

Journal of Biological Chemistry

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Aminoacyl-tRNA synthetase-containing complexes have been identified in different eukaryotes, and their existence has also been suggested in some Archaea. To investigate interactions involving aminoacyl-tRNA synthetases in Archaea, we undertook a yeast two-hybrid screen for interactions between Methanothermobacter thermautotrophicus proteins using prolyl-tRNA synthetase (ProRS) as the bait. Interacting proteins identified included components of methanogenesis, protein-modifying factors, and leucyl-tRNA synthetase (LeuRS). The association of ProRS with LeuRS was confirmed in vitro by native gel electrophoresis and size exclusion chromatography. Determination of the steady-state kinetics of tRNA Pro charging showed that the catalytic efficiency (k cat /K m ) of ProRS increased 5-fold in the complex with LeuRS compared with the free enzyme, whereas the K m for proline was unchanged. No significant changes in the steady-state kinetics of LeuRS aminoacylation were observed upon the addition of ProRS. These findings indicate that ProRS and LeuRS associate in M. thermautotrophicus and suggest that this interaction contributes to translational fidelity by enhancing tRNA aminoacylation by ProRS.Aminoacyl-tRNA synthetases (aaRSs) 1 are essential components of the translation process. Their cellular role is to ensure that individual tRNAs are attached to their cognate amino acid. Each aaRS specifically binds to a defined set of tRNAs and catalyzes the attachment of the amino acids to the tRNAs. Once synthesized, the aminoacyl-tRNAs function as substrates for ribosomal protein synthesis, thereby ensuring correct translation of the genetic code (1). In bacteria, aaRSs typically perform their role as individual enzymes, found either as monomers, homodimers, or homo-or heterotetramers. However, in eukaryotes several aminoacyl-tRNA synthetases exist in multienzyme complexes (2-4), and two different types have so far been found in mammalian cells. One is composed of only one aminoacyl-tRNA synthetase, valyl-tRNA synthetase, and EF-1H, the heavy form of translation elongation factor 1 (5). The complex is believed to contain seven subunits, two monomeric subunits of valyl-tRNA synthetase and the EF-1H subunits EF1-␣, -␤, -␥, and -␦ in the molar ratio 2:1:1:1. The second complex described is considerably larger and includes nine aminoacyl-tRNA synthetase activities. The complex is composed of isoleucyl-, leucyl-(LeuRS), prolyl-(ProRS), methionyl-, glutaminyl-, glutamyl-, lysyl-, arginyl-, and aspartyl-tRNA synthetases. Whereas most of the aaRSs are present in the complex as monomers, data have indicated that lysyltRNA synthetase and aspartyl-tRNA synthetase exist as dimers (3,6). In addition, the polypeptide carrying the ProRS activity is multifunctional in that the protein also comprises the catalytic domain and activity of glutamyl-tRNA synthetase (7). Three auxiliary proteins, p18, p38, and p43, are also part of the multisynthetase complex. Although the structural and functional significance of the complex still remains to be elu...

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Section: Discussionsupporting

confidence: 77%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Association between Archaeal Prolyl- and Leucyl-tRNA Synthetases Enhances tRNAPro Aminoacylation

Prætorius-Ibba¹,

Rogers²,

Samson³

et al. 2005

Journal of Biological Chemistry

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“…2B). The fluorescence intensity versus tRNA concentration was fit to a quadratic binding formula (see "Experimental Procedures"), yielding a K d of 0.16 Ϯ 0.04 M, in the range reported for other cognate synthetase-tRNA interactions (41,42).…”

Section: Resultsmentioning

confidence: 99%

The Homotetrameric Phosphoseryl-tRNA Synthetase from Methanosarcina mazei Exhibits Half-of-the-sites Activity

Hauenstein

Hou

Perona

2008

Journal of Biological Chemistry

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Synthesis of cysteinyl-tRNACys in methanogenic archaea proceeds by a two-step pathway in which tRNA Cys is first aminoacylated with phosphoserine by phosphoseryl-tRNA synthetase (SepRS). Characterization of SepRS from the mesophile Methanosarcina mazei by gel filtration and nondenaturing mass spectrometry shows that the native enzyme exists as an ␣4 tetramer when expressed at high levels in Escherichia coli. However, active site titrations monitored by ATP/PP i burst kinetics, together with analysis of tRNA binding stoichiometry by fluorescence spectroscopy, show that the tetrameric enzyme binds two tRNAs and that only two of the four chemically equivalent subunits catalyze formation of phosphoseryl adenylate. Therefore, the phenomenon of half-of-the-sites activity, previously described for synthesis of 1 mol of tyrosyl adenylate by the dimeric class I tyrosyl-tRNA synthetase, operates as well in this homotetrameric class II tRNA synthetase. Analysis of cognate and noncognate reactions by ATP/PP i and aminoacylation kinetics strongly suggests that SepRS is able to discriminate against the noncognate amino acids glutamate, serine, and phosphothreonine without the need for a separate hydrolytic editing site. tRNA Cys binding to SepRS also enhances the capacity of the enzyme to discriminate among amino acids, indicating the existence of functional connectivity between the tRNA and amino acid binding sites of the enzyme.

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“…This activity was assayed by using TLC to monitor the forward reaction by which γ-32 P-ATP is converted to 32 P-PP i upon synthesis of Cys-AMP 27 . To increase the sensitivity of detection, the labeled ATP substrate was kept at near saturating concentrations.…”

Section: A Stable Mutant Of Human Cysrs Lacking the C-terminal Extensionmentioning

confidence: 99%

Kinetic Quality Control of Anticodon Recognition by a Eukaryotic Aminoacyl-tRNA Synthetase

Liu

Gamper

Shtivelband

et al. 2007

Journal of Molecular Biology

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SummaryAminoacyl-tRNA synthetases are an ancient class of enzymes responsible for the matching of amino acids with anticodon sequences of tRNAs. Eukaryotic tRNA synthetases are often larger than their bacterial counterparts, and several mammalian enzymes use the additional domains to facilitate assembly into a multi-synthetase complex. Human cysteinyl-tRNA synthetase (CysRS) does not associate with the multi-synthetase complex, yet contains a eukaryotic-specific C-terminal extension that follows the tRNA anticodon-binding domain. Here we show by mutational and kinetic analysis that the C-terminal extension of human CysRS is used to selectively improve recognition and binding of the anticodon sequence, such that the specificity of anticodon recognition by human CysRS is higher than that of its bacterial counterparts. However, the improved anticodon recognition is achieved at the expense of a significantly slower rate in the aminoacylation reaction, suggesting a previously unrecognized kinetic quality control mechanism. This kinetic quality control reflects an evolutionary adaptation of some tRNA synthetases to improve the anticodon specificity of tRNA aminoacylation from bacteria to humans, possibly to accommodate concomitant changes in codon usage.

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Association of an Aminoacyl-tRNA Synthetase with a Putative Metabolic Protein in Archaea

Cited by 23 publications

References 47 publications

Association between Archaeal Prolyl- and Leucyl-tRNA Synthetases Enhances tRNAPro Aminoacylation

Association between Archaeal Prolyl- and Leucyl-tRNA Synthetases Enhances tRNAPro Aminoacylation

The Homotetrameric Phosphoseryl-tRNA Synthetase from Methanosarcina mazei Exhibits Half-of-the-sites Activity

Kinetic Quality Control of Anticodon Recognition by a Eukaryotic Aminoacyl-tRNA Synthetase

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