tRNA processing by protein-only versus RNA-based RNase P: kinetic analysis reveals mechanistic differences

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• RNase P enzymes are crucial for tRNA 5'-end maturation.
• In Arabidopsis thaliana, this function is performed by three homologous polypeptides: proteinaceous RNase P (PRORP) 1, 2, and 3.
• Understanding the catalytic mechanisms of PRORP is essential for comprehending tRNA processing in plants.

Purpose of the Study:

• To perform the first kinetic analysis of Arabidopsis thaliana PRORP enzymes.
• To investigate the substrate specificity and catalytic mechanism of PRORPs.
• To compare the mechanism of PRORP with bacterial RNA-based RNase P.

Main Methods:

• Single-turnover kinetic analysis of PRORP1.
• Assessment of PRORP sensitivity to Rp-phosphorothioate modification at the tRNA cleavage site.
• Evaluation of the impact of inosine modification on tRNA guanine residues.

Main Results:

• PRORP1 exhibited a specificity constant of 3×10(6) M(-1) min(-1).
• PRORP enzymes showed significantly less inhibition (factor of five) by Rp-phosphorothioate modification compared to bacterial RNase P (three orders of magnitude).
• Modifications to guanine residues had minor effects on PRORP processing, indicating non-critical interactions.

Conclusions:

• The catalytic mechanism of proteinaceous RNase P differs fundamentally from RNA-based bacterial RNase P.
• PRORP likely does not involve direct metal-ion coordination to the Rp substituent during catalysis.
• Productive PRORP-substrate interaction is not critically dependent on specific tRNA body modifications.