Abstract
Unlike other transfer RNAs (tRNA)-modifying enzymes from the SPOUT methyltransferase superfamily, the tRNA (Um34/Cm34) methyltransferase TrmL lacks the usual extension domain for tRNA binding and consists only of a SPOUT domain. Both the catalytic and tRNA recognition mechanisms of this enzyme remain elusive. By using tRNAs purified from an Escherichia coli strain with the TrmL gene deleted, we found that TrmL can independently catalyze the methyl transfer from S-adenosyl-L-methionine to and isoacceptors without the involvement of other tRNA-binding proteins. We have solved the crystal structures of TrmL in apo form and in complex with S-adenosyl-homocysteine and identified the cofactor binding site and a possible active site. Methyltransferase activity and tRNA-binding affinity of TrmL mutants were measured to identify residues important for tRNA binding of TrmL. Our results suggest that TrmL functions as a homodimer by using the conserved C-terminal half of the SPOUT domain for catalysis, whereas residues from the less-conserved N-terminal half of the other subunit participate in tRNA recognition.
Highlights
Posttranscriptional modifications of transfer RNAs are commonplace among the three kingdoms of life
Modified transfer RNAs (tRNA), dTrmL EctRNALCeAuA and dTrmL EctRNALUeAuA, containing all other natural modifications but the modification at the target position were purified after overproduction in E. coli JW3581-1 deleted TrmL gene
The results showed that EcTrmL-R20E, -K42E, -R43E, -R45E, -R46E, -R59E, -R104E and -R129E had dissociation constants (KD) of SAH comparable to EcTrmL, suggesting that the defects in methyltransferase activity were not caused by reduced SAH binding, but instead from reduced tRNA binding
Summary
Posttranscriptional modifications of transfer RNAs (tRNAs) are commonplace among the three kingdoms of life. Large amount of genes are involved in tRNA modification, between 1 and 10% of the genes in a given genome encode enzymes involved in tRNA modifications [1,2,3,4,5,6]. The genes involved in tRNA modification outnumber the genes encoding the actual tRNAs, which indicates an extremely important role for these enzymes [1]. The basic functions of tRNA modifications can be arranged into three classes [7]. Various other modifications affect the tRNA identity [14,15,16,17]. Besides directly affecting tRNA function, tRNA modifications have been shown to play regulatory roles such as responding to cellular stress, cancer or other diseases [18,19]
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