Isolation and characterization of the human tRNA-(N1G37) methyltransferase (TRM5) and comparison to the Escherichia coli TrmD protein.
A human TRM5 cDNA has been cloned and recombinant tRNA-N(1)G37 methyltransferase was produced. The recombinant enzyme methylates the N1 position of guanosine 37 (G37) in selected tRNA transcripts utilizing S-adenosyl methionine. The effects of RNA sequence and structure on the methylation reaction in comparison between the Escherichia coli TrmD and human TRM5 recombinant enzymes are presented. G37-methylation by TRM5 occurs regardless of the nature of the nucleotide at position 36. TRM5 also methylates inosine at position 37 unlike TrmD, which recognizes the G36pG37 motif preferentially and does not methylate inosine. New evidence is presented concerning TrmD showing that with some tRNA species, A at position 36 is also recognized. The TRM5 enzyme is sensitive to subtle changes in the tRNA-protein tertiary interaction leading to loss of activity. The TrmD enzyme is more tolerant of alterations in tRNA-protein tertiary interactions as long as the core tRNA structure and the G36pG37 are present. The TRM5 enzyme does not have an absolute requirement for magnesium ions, whereas TrmD requires magnesium to express activity. TRM5 demonstrates much higher affinity for substrates with K(m) values for tRNA that are nanomolar. TrmD has K(m) values for tRNA in the micromolar range. Recombinant TRM5 appears to function as a 60 772 Da monomer, while recombinant TrmD functions as a homodimer of 30 586 Da subunits. Bioinformatic analysis of the human TRM5 genomic locus (KIAA1393) have identified TRM5 homologues in eukaryotes and archaea; however, no significantly homologous regions were identified in any prokaryotes including the TrmD gene.
Brulé
Hervé
H
Department of Microbiology and Immunology, Medical College of Virginia Campus of Virginia Commonwealth University, Institute for Structural Biology and Drug Discovery, 800 East Leigh Street, Suite 212, Richmond, Virginia 23298, USA.
Elliott
Mark
M
Redlak
Maria
M
Zehner
Zendra E
ZE
Holmes
W Michael
WM
D001483:Base Sequence; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006151:Guanosine / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008745:Methylation; D011485:Protein Binding; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins; D013379:Substrate Specificity; D012359:tRNA Methyltransferases / Q000302:isolation & purification / Q000378:metabolism*
D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D006151:Guanosine; D012343:RNA, Transfer; C489297:TrmD protein, E coli; D012359:tRNA Methyltransferases; C489294:TRMT5 protein, human; D008274:Magnesium