7-Methylguanine specific tRNA-methyltransferase from Escherichia coli

Area of Science:

• Molecular Biology
• Enzymology
• Biochemistry

Background:

• Transfer RNA (tRNA) undergoes various post-transcriptional modifications essential for its function.
• 7-methylguanine (m7G) is a critical modification found in tRNA, influencing its structure and interactions.
• Specific enzymes are responsible for catalyzing these vital tRNA modifications.

Purpose of the Study:

• To purify and characterize a 7-methylguanine (m7G) specific tRNA methyltransferase from E. coli.
• To investigate the substrate specificity and catalytic activity of the purified enzyme.
• To elucidate the recognition mechanism of the enzyme for its tRNA substrate.

Main Methods:

• Affinity chromatography using Sepharose bound with normal E. coli tRNA for enzyme purification.
• Enzymatic assays to measure the formation of m7G in submethylated tRNA.
• Characterization of methyl group transfer from S-adenosyl-methionine.

Main Results:

• A tRNA methyltransferase was purified approximately 1000-fold from E. coli MRE 600.
• The purified enzyme exclusively catalyzed the formation of m7G in submethylated bulk tRNA of E. coli K12 met- rel-.
• The enzyme successfully transferred methyl groups to initiator tRNA of B. subtilis, forming 0.8 moles of m7G per mole of tRNA.

Conclusions:

• The purified enzyme is a specific tRNA methyltransferase responsible for m7G formation.
• The enzyme exhibits specificity towards submethylated bulk tRNA and initiator tRNA.
• It is proposed that the enzyme recognizes an unpaired guanylate residue in the extra arm of the tRNA.