Abstract
The human tRNA m(5)C methyltransferase is a potential target for anticancer drugs because it is a novel downstream target of the proto-oncogene myc, mediating Myc-induced cell proliferation. Sequence comparisons of RNA m(5)C methyltransferases indicate that the eukaryotic enzymes possess, in addition to a conserved catalytic domain, a large characteristic carboxyl-terminal extension. To gain insight into the function of this additional domain, the modular architecture of the yeast tRNA m(5)C methyltransferase orthologue, Trm4p, was studied. The yeast enzyme catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to carbon 5 of cytosine at different positions depending on the tRNAs. By limited proteolysis, Trm4p was shown to be composed of two domains that have been separately produced and purified. Here we demonstrate that the aminoterminal domain, encompassing the active site, binds tRNA with similar affinity as the whole enzyme but shows low catalytic efficiency. The carboxyl-terminal domain displays only weak affinity for tRNA. It is not required for m(5)C formation and does not appear to contribute to substrate specificity. However, it enhances considerably the catalytic efficiency of the amino-terminal domain.
Highlights
The methylations at different base positions and at the 2Ј-hydroxyl group of ribose are the most frequently encountered modifications in all RNAs
We demonstrate that the aminoterminal domain, encompassing the active site, binds tRNA with similar affinity as the whole enzyme but shows low catalytic efficiency
NTrm4p Is Catalytically Active whereas CTrm4p Is Not—To test the tRNA MTase activity of the different proteins and verify that the product of methyl transfer is m5C, a mature yeast tRNAPhe transcript, which is potentially modified solely at position 49, in contrast to its intron-containing precursor which is methylated at both positions 40 and 49 (Fig. 4A) [6], was internally labeled with [␣-32P]CTP, subjected to methylation, and hydrolyzed by nuclease P1
Summary
The methylations at different base positions and at the 2Ј-hydroxyl group of ribose are the most frequently encountered modifications in all RNAs. NTrm4p Has Similar Affinity for tRNA as the Whole Enzyme whereas CTrm4p Displays Only Weak Affinity for tRNA—The apparent dissociation constants (Kd) for an unmodified mature yeast tRNAPhe transcript of the different proteins were determined using the nitrocellulose-binding assay (Table 1; Fig. 3).
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