Reengineering CCA-adding enzymes to function as (U,G)- or dCdCdA-adding enzymes or poly(C,A) and poly(U,G)...

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• CCA-adding enzymes are essential RNA polymerases that synthesize the 3'-terminal CCA sequence of transfer RNA (tRNA).
• These enzymes utilize either a ribonucleoprotein (class I) or protein-only (class II) template to guide nucleotide incorporation.
• Understanding their mechanism is key to deciphering enzyme specificity and evolutionary diversity within the nucleotidyltransferase family.

Purpose of the Study:

• To investigate the role of protein-nucleic acid interactions in determining the substrate specificity of CCA-adding enzymes.
• To engineer CCA-adding enzymes with altered nucleotide incorporation capabilities by modifying the protein template interaction.

Main Methods:

• Structure-guided mutagenesis of the class II Bacillus stearothermophilus CCA-adding enzyme.
• Introduction of mutations to reverse hydrogen bond polarity between nucleobases and the protein template.
• Characterization of modified enzymes for altered nucleotide addition specificity.

Main Results:

• Transformed CCA-adding enzymes into (U,G)-adding, UU-adding, G-adding, poly(G), and poly(C,A) polymerases through targeted mutations.
• Engineered a dCdCdA-adding enzyme by mutating a key arginine residue involved in ribose interaction.
• Demonstrated that mutations in helix J can allosterically influence nucleotide binding site specificity, even at a distance.

Conclusions:

• Enzyme specificity in both class I and II CCA-adding enzymes is governed by a complex network of hydrogen bonds involving the protein, incoming nucleotide, and tRNA.
• Protein-RNA collaboration in template formation likely contributes to the evolutionary adaptability of nucleotidyltransferases.
• This study provides insights into enzyme engineering and the fundamental principles of molecular recognition in RNA-protein interactions.