tRNA recognition by a bacterial tRNA Xm32 modification enzyme from the SPOUT methyltransferase superfamily

TrmJ proteins from the SPOUT methyltransferase superfamily are tRNA Xm32 modification enzymes that occur in bacteria and archaea. Unlike archaeal TrmJ, bacterial TrmJ require full-length tRNA molecules as substrates. It remains unknown how bacterial TrmJs recognize substrate tRNAs and specifically c...

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Bibliographic Details
Published in:Nucleic acids research 2015-09, Vol.43 (15), p.7489-7503
Main Authors: Liu, Ru-Juan, Long, Tao, Zhou, Mi, Zhou, Xiao-Long, Wang, En-Duo
Format: Article
Language:English
Subjects:
Amino Acid Motifs
Archaea
Escherichia coli
Escherichia coli - enzymology
Escherichia coli Proteins - chemistry
Escherichia coli Proteins - metabolism
Methyltransferases - chemistry
Methyltransferases - metabolism
Models, Molecular
Nucleic Acid Conformation
Nucleic Acid Enzymes
Protein Binding
Protein Structure, Tertiary
RNA, Transfer - chemistry
RNA, Transfer - metabolism
S-Adenosylhomocysteine - chemistry
S-Adenosylhomocysteine - metabolism
tRNA Methyltransferases - chemistry
tRNA Methyltransferases - metabolism
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Summary:TrmJ proteins from the SPOUT methyltransferase superfamily are tRNA Xm32 modification enzymes that occur in bacteria and archaea. Unlike archaeal TrmJ, bacterial TrmJ require full-length tRNA molecules as substrates. It remains unknown how bacterial TrmJs recognize substrate tRNAs and specifically catalyze a 2'-O modification at ribose 32. Herein, we demonstrate that all six Escherichia coli (Ec) tRNAs with 2'-O-methylated nucleosides at position 32 are substrates of EcTrmJ, and we show that the elbow region of tRNA, but not the amino acid acceptor stem, is needed for the methylation reaction. Our crystallographic study reveals that full-length EcTrmJ forms an unusual dimer in the asymmetric unit, with both the catalytic SPOUT domain and C-terminal extension forming separate dimeric associations. Based on these findings, we used electrophoretic mobility shift assay, isothermal titration calorimetry and enzymatic methods to identify amino acids within EcTrmJ that are involved in tRNA binding. We found that tRNA recognition by EcTrmJ involves the cooperative influences of conserved residues from both the SPOUT and extensional domains, and that this process is regulated by the flexible hinge region that connects these two domains.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkv745