PMID:15850396
| Citation |
Watts, JM, Gabruzsk, J and Holmes, WM (2005) Ligand-mediated anticodon conformational changes occur during tRNA methylation by a TrmD methyltransferase. Biochemistry 44:6629-39 |
|---|---|
| Abstract |
Orthologs of TrmD, G37 tRNA methyltransferases, have been analyzed with regard to post-tRNA binding events required to move the residue G37 in proximity to bound AdoMet for catalysis. This was approached initially by probing tRNA with T2 nuclease or Pb acetate in the presence, then absence, of Escherichia coli TrmD protein. Cleavage patterns clearly show that portions of the anticodon loop phosphodiester backbone are protected from cleavage only in the presence of sinefungin, a potent AdoMet analogue. This demonstrates that there must be considerable movement of the loop region and/or protein as the AdoMet site is occupied. Florescence energy transfer experiments were employed to better assess the movement of the G37 and G36 base residues in response to occupancy of the AdoMet site. When the Streptococcus pneumoniae TrmD protein was bound to synthetic tRNA(1)(Leu) substituted with 2-aminopurine at positions 36 and 37, fluorescence energy transfer analysis showed that a decrease in 2-aminopurine fluorescence occurs only when AdoMet is present. Taken together, these results suggest that the base to be methylated by the TrmD protein is mobilized into the active center after tRNA binding only when the AdoMet site is occupied. |
| Links |
PubMed Online version:10.1021/bi0481038 |
| Keywords |
Adenosine/analogs & derivatives; Adenosine/chemistry; Amino Acid Sequence; Anticodon/chemistry; Anticodon/metabolism; Base Sequence; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Fluorescence Resonance Energy Transfer; Ligands; Methylation; Models, Molecular; Molecular Sequence Data; Nucleic Acid Conformation; Protein Conformation; Protein Footprinting; RNA, Transfer, Leu/chemistry; RNA, Transfer, Leu/genetics; RNA, Transfer, Leu/metabolism; RNA-Binding Proteins/chemistry; RNA-Binding Proteins/genetics; RNA-Binding Proteins/metabolism; S-Adenosylmethionine/chemistry; S-Adenosylmethionine/metabolism; Sequence Alignment; Thermotoga maritima/enzymology; Thermotoga maritima/genetics; tRNA Methyltransferases/chemistry; tRNA Methyltransferases/genetics; tRNA Methyltransferases/metabolism |
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