The gene for mRNA 5′-cap methyltransferase: identification of additional residues essential for catalysis

The GenBank accession numbers for the nucleotide sequence of and are AB020965 and AB020966, respectively.

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Abstract

The 5′-cap structure of eukaryotic mRNA is methylated at the terminal guanosine by RNA (guanine- -)-methyltransferase (cap MTase). () and human (also called ) genes are responsible for this enzyme. The homologue was cloned from the pathogenic fungus and named (). When expressed as a fusion with glutathione -transferase (GST), CaAbd1p displayed cap MTase activity and rescued Δ null mutants, indicating that specifies an active cap MTase. Although the human cap MTase binds to the human capping enzyme (Hce1p), which possesses both mRNA guanylyltransferase (mRNA GTase) and mRNA 5′-triphosphatase (mRNA TPase) activities, yeast two-hybrid analysis demonstrated that in yeast neither mRNA GTase nor mRNA TPase physically interacted with the Abd1 protein. Comparison of the amino acid sequences of known and putative cap MTases revealed a highly conserved amino acid sequence motif, Phe/Val-Leu-Asp/Glu-Leu/Met-Xaa-Cys-Gly-Lys-Gly-Gly-Asp-Leu-Xaa-Lys, which encompasses the sequence motif characteristic of divergent methyltransferases. Mutations in CaAbd1p of leucine at the second and the twelfth positions (so far uncharacterized) to alanine severely diminished the enzyme activity and the functionality , whereas those of leucine at the fourth, cysteine at the sixth, lysine at the eighth, and glycine at the tenth positions did not. Furthermore, valine substitution for the twelfth, but not for the second, leucine in that motif abolished the activity and functionality of CaAbd1p. Thus, it appears that leucine at the second and the twelfth positions in the motif, together with a previously identified acidic residue in the third, glycine at the sixth and glutamic acid at the eleventh positions, play important roles in the catalysis, and that side chain length is crucial for the activity at the twelfth position in the motif.

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1999-11-01
2024-03-28
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