Conformational and Functional Divergence of RnpA Protein Subfamilies in Bacterial Ribonucleoprotein RNase P

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Bacterial ribonuclease P (RNase P) is a vital ribonucleoprotein enzyme.
• It consists of a catalytic RNA subunit (P RNA) and a protein cofactor (RnpA).
• RnpA is crucial for catalysis and precursor tRNA (ptRNA) binding, but sequence variation effects are unclear.

Purpose of the Study:

• To investigate how sequence variation in bacterial RnpA proteins influences enzyme folding, dynamics, and catalytic function.
• To understand the relationship between RnpA subfamilies and P RNA types (A and B).
• To elucidate the specific contributions of RnpA to substrate binding and catalytic activation.

Main Methods:

• Sequence similarity network (SSN) analysis of over 1800 RnpA sequences.
• Computational and biophysical studies (e.g., conformational dynamics, thermal stability).
• Enzyme kinetics (kcat, KM) and reconstitution assays with cognate and noncognate subunits.

Main Results:

• Identified two major RnpA subfamilies: RnpA-1 (Bacilli-specific, Type B P RNA) and RnpA-2 (broader, Type A P RNA).
• RnpA-1 proteins show greater dynamics and lower stability than RnpA-2 proteins.
• Both RnpA families effectively enhance ptRNA binding, but Type B RNase Ps have higher kcat, while Type A enzymes are limited by precatalytic steps.

Conclusions:

• RnpA sequence variation leads to distinct structural and dynamic properties, influencing RNase P enzyme function.
• RnpA identity critically impacts substrate binding and catalytic steps, with different rate-limiting steps for Type A and B enzymes.
• These findings provide a basis for understanding RnpA function and developing targeted inhibitors.