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1.  SAXS analysis of the tRNA-modifying enzyme complex MnmE/MnmG reveals a novel interaction mode and GTP-induced oligomerization 
Nucleic Acids Research  2014;42(9):5978-5992.
Transfer ribonucleic acid (tRNA) modifications, especially at the wobble position, are crucial for proper and efficient protein translation. MnmE and MnmG form a protein complex that is implicated in the carboxymethylaminomethyl modification of wobble uridine (cmnm5U34) of certain tRNAs. MnmE is a G protein activated by dimerization (GAD), and active guanosine-5'-triphosphate (GTP) hydrolysis is required for the tRNA modification to occur. Although crystal structures of MnmE and MnmG are available, the structure of the MnmE/MnmG complex (MnmEG) and the nature of the nucleotide-induced conformational changes and their relevance for the tRNA modification reaction remain unknown. In this study, we mainly used small-angle X-ray scattering to characterize these conformational changes in solution and to unravel the mode of interaction between MnmE, MnmG and tRNA. In the nucleotide-free state MnmE and MnmG form an unanticipated asymmetric α2β2 complex. Unexpectedly, GTP binding promotes further oligomerization of the MnmEG complex leading to an α4β2 complex. The transition from the α2β2 to the α4β2 complex is fast, reversible and coupled to GTP binding and hydrolysis. We propose a model in which the nucleotide-induced changes in conformation and oligomerization of MnmEG form an integral part of the tRNA modification reaction cycle.
doi:10.1093/nar/gku213
PMCID: PMC4027165  PMID: 24634441
2.  Crystallization and preliminary X-ray crystallographic analysis of putative tRNA-modification enzymes from Pyrococcus furiosus and Thermus thermophilus  
Two orthologous putative tRNA methyltransferases from P. furiosus and T. thermophilus have been expressed, purified and crystallized. X-ray diffraction data were collected to 2.2 and 2.05 Å, respectively.
Methyltransferases form a major class of tRNA-modifying enzymes that are needed for the proper functioning of tRNA. Here, the expression, purification and crystallization of two related putative tRNA methyltransferases from two kingdoms of life are reported. The protein encoded by the gene pf1002 from the archaeon Pyrococcus furiosus was crystallized in the monoclinic space group P21. A complete data set was collected to 2.2 Å resolution. The protein encoded by the gene ttc1157 from the eubacterium Thermus thermophilus was crystallized in the trigonal space group P3221. A complete data set was collected to 2.05 Å resolution.
doi:10.1107/S1744309111036347
PMCID: PMC3212469  PMID: 22102250
tRNA; methyltransferases; ttc1157; pf1002
3.  Crystal structures of the tRNA:m2G6 methyltransferase Trm14/TrmN from two domains of life 
Nucleic Acids Research  2012;40(11):5149-5161.
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 tRNAPhe at guanosine 6 in the tRNA acceptor stem. Here, we report the first X-ray crystal structures of the tRNA m2G6 (N2-methylguanosine) MTase TTCTrmN from Thermus thermophilus and its ortholog PfTrm14 from Pyrococcus furiosus. Structures of PfTrm14 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. TTCTrmN and PfTrm14 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 tRNAPhe of T. thermophilus and via site-directed mutagenesis.
doi:10.1093/nar/gks163
PMCID: PMC3367198  PMID: 22362751

Results 1-3 (3)