Multiple substrate binding sites in the ribozyme from Bacillus subtilis RNase P

Area of Science:

• Biochemistry
• Molecular Biology
• RNA Biology

Background:

• The Bacillus subtilis RNase P (P RNA) ribozyme is known to process tRNA substrates by recognizing specific RNA structural motifs.
• Understanding the substrate specificity of ribozymes is crucial for elucidating their biological functions and potential applications.

Purpose of the Study:

• To investigate if P RNA can interact with RNA substrates beyond canonical tRNA structures.
• To identify novel RNA substrates and binding sites for P RNA using in vitro selection.

Main Methods:

• In vitro selection was employed using a modified P RNA ribozyme lacking the T stem-loop binding site.
• Nine rounds of selection were performed to isolate RNA molecules with altered binding properties.
• Biochemical characterization was used to analyze the interaction of the selected RNA with both the modified and full-length P RNA.

Main Results:

• A single, novel RNA substrate was successfully isolated after selection.
• This selected RNA is a competent substrate for both the modified and full-length P RNA.
• Biochemical assays revealed that the selected substrate interacts with P RNA at a distinct site compared to tRNA substrates.
• A self-cleaving RNA with unique characteristics was also identified during the selection process.

Conclusions:

• Biological ribozymes, like P RNA, possess the capacity to bind and process diverse RNA substrates through alternative binding sites.
• This finding supports an alternate binding site model, proposing a mechanism for the evolution of ribozymes to accommodate a broader range of RNA structures.