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Crystal structures of the tRNA:m2G6 methyltransferase Trm14/TrmN from two domains of life.

ABSTRACT: Methyltransferases (MTases) form a major class of tRNA-modifying enzymes needed for the proper functioning of tRNA. Recently, RNA MTases from the TrmN/Trm14 family that are present in Archaea, Bacteria and Eukaryota have been shown to specifically modify tRNA(Phe) at guanosine 6 in the tRNA acceptor stem. Here, we report the first X-ray crystal structures of the tRNA m(2)G6 (N(2)-methylguanosine) MTase (TTC)TrmN from Thermus thermophilus and its ortholog (Pf)Trm14 from Pyrococcus furiosus. Structures of (Pf)Trm14 were solved in complex with the methyl donor S-adenosyl-l-methionine (SAM or AdoMet), as well as the reaction product S-adenosyl-homocysteine (SAH or AdoHcy) and the inhibitor sinefungin. (TTC)TrmN and (Pf)Trm14 consist of an N-terminal THUMP domain fused to a catalytic Rossmann-fold MTase (RFM) domain. These results represent the first crystallographic structure analysis of proteins containing both THUMP and RFM domain, and hence provide further insight in the contribution of the THUMP domain in tRNA recognition and catalysis. Electrostatics and conservation calculations suggest a main tRNA binding surface in a groove between the THUMP domain and the MTase domain. This is further supported by a docking model of TrmN in complex with tRNA(Phe) of T. thermophilus and via site-directed mutagenesis.

SUBMITTER: Fislage M

PROVIDER: S-EPMC3367198 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Crystal structures of the tRNA:m2G6 methyltransferase Trm14/TrmN from two domains of life.

Fislage Marcus M Roovers Martine M Tuszynska Irina I Bujnicki Janusz M JM Droogmans Louis L Versées Wim W

Nucleic acids research 20120222 11

Methyltransferases (MTases) form a major class of tRNA-modifying enzymes needed for the proper functioning of tRNA. Recently, RNA MTases from the TrmN/Trm14 family that are present in Archaea, Bacteria and Eukaryota have been shown to specifically modify tRNA(Phe) at guanosine 6 in the tRNA acceptor stem. Here, we report the first X-ray crystal structures of the tRNA m(2)G6 (N(2)-methylguanosine) MTase (TTC)TrmN from Thermus thermophilus and its ortholog (Pf)Trm14 from Pyrococcus furiosus. Struc ...[more]

PMID: 22362751

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Project description:Tryptophanyl-tRNA synthetase (TrpRS) is an essential enzyme that is recognizably conserved across all forms of life. It is responsible for activating and attaching tryptophan to a cognate tRNA(Trp) molecule for use in protein synthesis. In some eukaryotes this original core function has been supplemented or modified through the addition of extra domains or the expression of variant TrpRS isoforms. The three TrpRS structures from pathogenic protozoa described here represent three illustrations of this malleability in eukaryotes. The Cryptosporidium parvum genome contains a single TrpRS gene, which codes for an N-terminal domain of uncertain function in addition to the conserved core TrpRS domains. Sequence analysis indicates that this extra domain, conserved among several apicomplexans, is related to the editing domain of some AlaRS and ThrRS. The C. parvum enzyme remains fully active in charging tRNA(Trp) after truncation of this extra domain. The crystal structure of the active, truncated enzyme is presented here at 2.4Å resolution. The Trypanosoma brucei genome contains separate cytosolic and mitochondrial isoforms of TrpRS that have diverged in their respective tRNA recognition domains. The crystal structure of the T. brucei cytosolic isoform is presented here at 2.8Å resolution. The Entamoeba histolytica genome contains three sequences that appear to be TrpRS homologs. However one of these, whose structure is presented here at 3.0Å resolution, has lost the active site motifs characteristic of the Class I aminoacyl-tRNA synthetase catalytic domain while retaining the conserved features of a fully formed tRNA(Trp) recognition domain. The biological function of this variant E. histolytica TrpRS remains unknown, but, on the basis of a completely conserved tRNA recognition region and evidence for ATP but not tryptophan binding, it is tempting to speculate that it may perform an editing function. Together with a previously reported structure of an unusual TrpRS from Giardia, these protozoan structures broaden our perspective on the extent of structural variation found in eukaryotic TrpRS homologs.

Dataset Information

Crystal structures of the tRNA:m2G6 methyltransferase Trm14/TrmN from two domains of life.

Publications

Crystal structures of the tRNA:m2G6 methyltransferase Trm14/TrmN from two domains of life.

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