Structural basis of reverse nucleotide polymerization.

Area of Science:

• Molecular Biology
• Enzymology
• Structural Biology

Background:

• Nucleotide polymerization is fundamentally understood to occur in the 5"-3" direction, a principle central to molecular biology.
• Processes like DNA replication, transcription, and reverse transcription adhere to this forward directionality, despite simpler alternatives.
• Exceptions to this rule exist, such as the 3"-5" polymerization catalyzed by tRNA(His) guanylyltransferase.

Purpose of the Study:

• To elucidate the structural mechanisms underlying reverse (3"-5") nucleotide polymerization.
• To investigate the role of Candida albicans tRNA(His) guanylyltransferase in this process.

Main Methods:

• Determined the complex structure of Candida albicans tRNA(His) guanylyltransferase bound to tRNA(His).
• Employed structural, biochemical, and phylogenetic analyses.
• Analyzed the orientation of nucleotide substrate approach and enzyme domain positioning.

Main Results:

• The structure reveals that reverse polymerization is dictated by the opposite (180° flip) orientation of the nucleotide substrate's approach compared to canonical 5"-3" polymerases.
• Finger domains are positioned on opposing sides of the core palm domain, facilitating the reverse directionality.
• Phylogenetic data suggest reverse polymerization is an ancient process, mirroring forward polymerization.

Conclusions:

• The directionality of nucleotide polymerization is determined by substrate approach orientation and enzyme architecture.
• Reverse polymerization, catalyzed by tRNA(His) guanylyltransferase, represents an evolutionarily early mechanism.
• This finding offers insights into the diversity of polymerization mechanisms beyond the canonical 5"-3" direction.