3'-5' tRNAHis guanylyltransferase in bacteria

Area of Science:

• Molecular Biology
• Enzymology
• Microbial Genetics

Background:

• Histidine-specific transfer RNA (tRNA(His)) identity is crucial for protein synthesis.
• This identity is often established by a unique guanosine at position -1.
• The enzyme tRNA(His) guanylyltransferase (Thg1) is known to catalyze this modification in eukaryotes and archaea.

Purpose of the Study:

• To investigate the presence and function of Thg1 in bacteria.
• To compare the enzymatic mechanism of bacterial Thg1 with its eukaryotic and archaeal counterparts.
• To assess the in vivo activity and substrate specificity of bacterial Thg1.

Main Methods:

• Recombinant expression and in vitro activity assays of bacterial Thg1 enzymes from Bacillus thuringiensis and Myxococcus xanthus.
• Biochemical characterization of archaeal Thg1 enzymes.
• Complementation studies using a yeast thg1 knockout strain.

Main Results:

• Thg1 activity was demonstrated in vitro for bacterial enzymes.
• Prokaryotic Thg1 utilizes a distinct reaction mechanism that does not require ATP.
• Bacterial Thg1 successfully complemented a yeast thg1 knockout strain in vivo, indicating functional activity.
• Bacterial Thg1 exhibits relaxed recognition of the tRNA discriminator base.

Conclusions:

• Thg1 is present and functional in bacteria, expanding its known phylogenetic distribution.
• Bacterial Thg1 represents a distinct class of enzymes with a unique ATP-independent catalytic mechanism.
• The findings suggest evolutionary divergence in tRNA modification pathways across different domains of life.