Mutational analysis of yeast mRNA capping enzyme

Area of Science:

• Molecular Biology
• Enzymology
• Eukaryotic Gene Expression

Background:

• RNA guanylyltransferase (capping enzyme) is crucial for mRNA processing.
• The enzyme forms a covalent GMP-enzyme intermediate.
• The CEG1 gene product in Saccharomyces cerevisiae is a 52-kDa polypeptide.

Purpose of the Study:

• To investigate the role of the conserved Lys70-Thr-Asp-Gly motif in yeast RNA guanylyltransferase function.
• To determine the essentiality of specific residues within this motif for enzyme activity and cell viability.

Main Methods:

• Expression of the CEG1 protein in Escherichia coli.
• Site-directed mutagenesis of the CEG1 gene, specifically targeting Lys70, Thr71, Asp72, and Gly73.
• In vitro enzyme assays to assess guanylyltransferase activity.
• Analysis of yeast viability upon expression of mutated CEG1 alleles.

Main Results:

• Mutations replacing Lys70 or Gly73 with alanine abolished enzyme-guanylate formation in vitro.
• Alanine substitutions at Thr71 and Asp72 reduced enzyme activity but did not abrogate it.
• The K70A and G73A mutations were lethal in yeast, while T71A and D72A mutations allowed cell viability.

Conclusions:

• Lysine 70 is identified as the active site lysine residue in yeast RNA guanylyltransferase.
• mRNA capping is an essential function for eukaryotic cell survival.
• The conserved KXDG motif plays a critical role in the catalytic mechanism of RNA capping enzymes.