Human trans-editing enzyme displays tRNA acceptor stem specificity and relaxed amino acid selectivity

AbstractAccurate translation of genetic information into proteins is vital for cell sustainability. ProXp-ala prevents proteome-wide Pro-to-Ala mutations by hydrolyzing misacylated Ala-tRNAPro, which is synthesized by prolyl-tRNA synthetase (ProRS). Bacterial ProXp-ala was previously shown to combine a size-based exclusion mechanism with conformational and chemical selection for the recognition of the alanyl moiety, while tRNAPro is selected via recognition of tRNA acceptor stem elements G72 and A73. The identity of these critical bases changed during evolution with eukaryotic cytosolic tRNAPro possessing a cytosine at the corresponding positions. The mechanism by which eukaryotic ProXp-ala adapted to these changes remains unknown. In this work, recognition of the aminoacyl moiety and tRNA acceptor stem by human (Hs) ProXp-ala was examined. Enzymatic assays revealed that Hs ProXp-ala requires C72 and C73 in the context of Hs cytosolic tRNAPro for efficient deacylation of mischarged Ala-tRNAPro. The strong dependence on these bases prevents cross-species deacylation of bacterial Ala-tRNAPro or of Hs mitochondrial Ala-tRNAPro by the human enzyme. Similar to the bacterial enzyme, Hs ProXp-ala showed strong tRNA acceptor-stem recognition but differed in its amino acid specificity profile relative to bacterial ProXp-ala. Changes at conserved residues in both the Hs and bacterial ProXp-ala substrate binding pockets modulated this specificity. These results illustrate how the mechanism of substrate selection diverged during the evolution of the ProXp-ala family and provides the first example of a trans-editing domain whose specificity evolved to adapt to changes in its tRNA substrate.

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Human trans-editing enzyme displays tRNA acceptor-stem specificity and relaxed amino acid selectivity

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.015981 ◽

2020 ◽

Vol 295 (48) ◽

pp. 16180-16190

Author(s):

Oscar Vargas-Rodriguez ◽

Marina Bakhtina ◽

Daniel McGowan ◽

Jawad Abid ◽

Yuki Goto ◽

...

Keyword(s):

Amino Acid ◽

Homo Sapiens ◽

Strong Dependence ◽

Trna Synthetase ◽

Substrate Selection ◽

Acceptor Stem ◽

Bacterial Enzyme ◽

Editing Domain ◽

Exclusion Mechanism ◽

Trna Acceptor

Accurate translation of genetic information into proteins is vital for cell sustainability. ProXp-ala prevents proteome-wide Pro-to-Ala mutations by hydrolyzing misacylated Ala-tRNAPro, which is synthesized by prolyl-tRNA synthetase. Bacterial ProXp-ala was previously shown to combine a size-based exclusion mechanism with conformational and chemical selection for the recognition of the alanyl moiety, whereas tRNAPro is selected via recognition of tRNA acceptor-stem elements G72 and A73. The identity of these critical bases changed during evolution with eukaryotic cytosolic tRNAPro possessing a cytosine at the corresponding positions. The mechanism by which eukaryotic ProXp-ala adapted to these changes remains unknown. In this work, recognition of the aminoacyl moiety and tRNA acceptor stem by human (Homo sapiens, or Hs) ProXp-ala was examined. Enzymatic assays revealed that Hs ProXp-ala requires C72 and C73 in the context of Hs cytosolic tRNAPro for efficient deacylation of mischarged Ala-tRNAPro. The strong dependence on these bases prevents cross-species deacylation of bacterial Ala-tRNAPro or of Hs mitochondrial Ala-tRNAPro by the human enzyme. Similar to the bacterial enzyme, Hs ProXp-ala showed strong tRNA acceptor-stem recognition but differed in its amino acid specificity profile relative to bacterial ProXp-ala. Changes at conserved residues in both the Hs and bacterial ProXp-ala substrate-binding pockets modulated this specificity. These results illustrate how the mechanism of substrate selection diverged during the evolution of the ProXp-ala family, providing the first example of a trans-editing domain whose specificity evolved to adapt to changes in its tRNA substrate.

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Structural and mutational studies of the amino acid-editing domain from archaeal/eukaryal phenylalanyl-tRNA synthetase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0603182103 ◽

2006 ◽

Vol 103 (40) ◽

pp. 14744-14749 ◽

Cited By ~ 26

Author(s):

H. M. Sasaki ◽

S.-i. Sekine ◽

T. Sengoku ◽

R. Fukunaga ◽

M. Hattori ◽

...

Keyword(s):

Amino Acid ◽

Trna Synthetase ◽

Amino Acid Editing ◽

Editing Domain

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Role of Zinc in tRNA-acceptor Stem Binding by Glutamyl-tRNA Synthetase FromE.coli: A Molecular Modeling Study

Journal of Biomolecular Structure and Dynamics ◽

10.1080/07391102.1997.10508941 ◽

1997 ◽

Vol 15 (1) ◽

pp. 19-25

Author(s):

Asim K. Bothra ◽

Siddhartha Roy ◽

Chhabinath Mandal ◽

Chaitali Mukhophadhyay

Keyword(s):

Molecular Modeling ◽

Trna Synthetase ◽

Acceptor Stem ◽

Molecular Modeling Study ◽

Trna Acceptor ◽

Modeling Study

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Human cytoplasmic ProX edits mischarged tRNAPro with amino acid but not tRNA specificity

Biochemical Journal ◽

10.1042/bj20121493 ◽

2013 ◽

Vol 450 (1) ◽

pp. 243-252 ◽

Cited By ~ 12

Author(s):

Liang-Liang Ruan ◽

Xiao-Long Zhou ◽

Min Tan ◽

En-Duo Wang

Keyword(s):

Amino Acid ◽

Protein Biosynthesis ◽

Trna Synthetase ◽

Main Body ◽

Amino Acid Residues ◽

Homologous Proteins ◽

Gene Encoding ◽

Trna Synthetases ◽

Editing Domain ◽

First Time

aaRSs (aminoacyl-tRNA synthetases) are responsible for ensuring the fidelity of the genetic code translation by accurately linking a particular amino acid to its cognate tRNA isoacceptor. To ensure accuracy of protein biosynthesis, some aaRSs have evolved an editing process to remove mischarged tRNA. The hydrolysis of the mischarged tRNA usually occurs in an editing domain, which is inserted into or appended to the main body of the aaRS. In addition, autonomous, editing domain-homologous proteins can also trans-edit mischarged tRNA in concert or in compensating for the editing function of its corresponding aaRS. The freestanding ProX is a homologue of the editing domain of bacterial ProRS (prolyl-tRNA synthetase). In the present study, we cloned for the first time a gene encoding HsProX (human cytoplasmic ProX) and purified the expressed recombinant protein. The catalytic specificity of HsProX for non-cognate amino acids and identity elements on tRNAPro for editing were also investigated. We found that HsProX could deacylate mischarged Ala-tRNAPro, but not Cys-HstRNAUGGPro, and specifically targeted the alanine moiety of Ala-tRNAPro. The importance of the CCA76 end of the tRNA for deacylation activity and key amino acid residues in HsProX for its editing function were also identified.

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Structural Basis for Non-cognate Amino Acid Discrimination by the Valyl-tRNA Synthetase Editing Domain

Journal of Biological Chemistry ◽

10.1074/jbc.m502668200 ◽

2005 ◽

Vol 280 (33) ◽

pp. 29937-29945 ◽

Cited By ~ 31

Author(s):

Ryuya Fukunaga ◽

Shigeyuki Yokoyama

Keyword(s):

Amino Acid ◽

Trna Synthetase ◽

Structural Basis ◽

Editing Domain

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tRNA acceptor-stem and anticodon bases embed separate features of amino acid chemistry

RNA Biology ◽

10.1080/15476286.2015.1112488 ◽

2015 ◽

Vol 13 (2) ◽

pp. 145-151 ◽

Cited By ~ 17

Author(s):

Charles W. Carter ◽

Richard Wolfenden

Keyword(s):

Amino Acid ◽

Acceptor Stem ◽

Trna Acceptor

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Indirect Routes to Aminoacyl-tRNA: The Diversity of Prokaryotic Cysteine Encoding Systems

Frontiers in Genetics ◽

10.3389/fgene.2021.794509 ◽

2022 ◽

Vol 12 ◽

Author(s):

Takahito Mukai ◽

Kazuaki Amikura ◽

Xian Fu ◽

Dieter Söll ◽

Ana Crnković

Keyword(s):

Amino Acid ◽

Genetic Code ◽

Elongation Factor ◽

Methanogenic Archaea ◽

Bioinformatic Analysis ◽

Trna Synthetase ◽

Indirect Pathway ◽

Gradual Loss ◽

Editing Domain ◽

Diverse Groups

Universally present aminoacyl-tRNA synthetases (aaRSs) stringently recognize their cognate tRNAs and acylate them with one of the proteinogenic amino acids. However, some organisms possess aaRSs that deviate from the accurate translation of the genetic code and exhibit relaxed specificity toward their tRNA and/or amino acid substrates. Typically, these aaRSs are part of an indirect pathway in which multiple enzymes participate in the formation of the correct aminoacyl-tRNA product. The indirect cysteine (Cys)-tRNA pathway, originally thought to be restricted to methanogenic archaea, uses the unique O-phosphoseryl-tRNA synthetase (SepRS), which acylates the non-proteinogenic amino acid O-phosphoserine (Sep) onto tRNACys. Together with Sep-tRNA:Cys-tRNA synthase (SepCysS) and the adapter protein SepCysE, SepRS forms a transsulfursome complex responsible for shuttling Sep-tRNACys to SepCysS for conversion of the tRNA-bound Sep to Cys. Here, we report a comprehensive bioinformatic analysis of the diversity of indirect Cys encoding systems. These systems are present in more diverse groups of bacteria and archaea than previously known. Given the occurrence and distribution of some genes consistently flanking SepRS, it is likely that this gene was part of an ancient operon that suffered a gradual loss of its original components. Newly identified bacterial SepRS sequences strengthen the suggestion that this lineage of enzymes may not rely on the m1G37 identity determinant in tRNA. Some bacterial SepRSs possess an N-terminal fusion resembling a threonyl-tRNA synthetase editing domain, which interestingly is frequently observed in the vicinity of archaeal SepCysS genes. We also found several highly degenerate SepRS genes that likely have altered amino acid specificity. Cross-analysis of selenocysteine (Sec)-utilizing traits confirmed the co-occurrence of SepCysE and the Sec-utilizing machinery in archaea, but also identified an unusual O-phosphoseryl-tRNASec kinase fusion with an archaeal Sec elongation factor in some lineages, where it may serve in place of SepCysE to prevent crosstalk between the two minor aminoacylation systems. These results shed new light on the variations in SepRS and SepCysS enzymes that may reflect adaptation to lifestyle and habitat, and provide new information on the evolution of the genetic code.

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Homologous trans-editing factors with broad tRNA specificity prevent mistranslation caused by serine/threonine misactivation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1423664112 ◽

2015 ◽

Vol 112 (19) ◽

pp. 6027-6032 ◽

Cited By ~ 19

Author(s):

Ziwei Liu ◽

Oscar Vargas-Rodriguez ◽

Yuki Goto ◽

Eva Maria Novoa ◽

Lluís Ribas de Pouplana ◽

...

Keyword(s):

Amino Acid ◽

Trna Synthetase ◽

Control Mechanisms ◽

Free Standing ◽

Selective Growth Advantage ◽

Editing Domain ◽

Multifunctional Enzymes ◽

Species Specific

Aminoacyl-tRNA synthetases (ARSs) establish the rules of the genetic code, whereby each amino acid is attached to a cognate tRNA. Errors in this process lead to mistranslation, which can be toxic to cells. The selective forces exerted by species-specific requirements and environmental conditions potentially shape quality-control mechanisms that serve to prevent mistranslation. A family of editing factors that are homologous to the editing domain of bacterial prolyl-tRNA synthetase includes the previously characterized trans-editing factors ProXp-ala and YbaK, which clear Ala-tRNAPro and Cys-tRNAPro, respectively, and three additional homologs of unknown function, ProXp-x, ProXp-y, and ProXp-z. We performed an in vivo screen of 230 conditions in which an Escherichia coli proXp-y deletion strain was grown in the presence of elevated levels of amino acids and specific ARSs. This screen, together with the results of in vitro deacylation assays, revealed Ser- and Thr-tRNA deacylase function for this homolog. A similar activity was demonstrated for Bordetella parapertussis ProXp-z in vitro. These proteins, now renamed “ProXp-ST1” and “ProXp-ST2,” respectively, recognize multiple tRNAs as substrates. Taken together, our data suggest that these free-standing editing domains have the ability to prevent mistranslation errors caused by a number of ARSs, including lysyl-tRNA synthetase, threonyl-tRNA synthetase, seryl-tRNA synthetase, and alanyl-tRNA synthetase. The expression of these multifunctional enzymes is likely to provide a selective growth advantage to organisms subjected to environmental stresses and other conditions that alter the amino acid pool.

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