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Characterization of functionally active subribosomal particles from Thermus aquaticus
P Khaitovich1, A S Mankin, R Green
1Center for Pharmaceutical Biotechnology, m/c 870, University of Illinois, 900 South Ashland Avenue, Chicago, IL 60607, USA.
Summary
The Thermus aquaticus peptidyl transferase center retains activity even with many proteins removed. Only 23S rRNA and proteins L2 and L3 appear essential for this core function.
Area of Science:
- Molecular Biology
- Biochemistry
- Structural Biology
Background:
- Ribosomes are crucial for protein synthesis.
- The peptidyl transferase center (PTC) is responsible for peptide bond formation.
- Understanding the minimal components of the PTC is key to deciphering its function.
Purpose of the Study:
- To determine the minimum number of macromolecular components required for peptidyl transferase activity.
- To identify the essential ribosomal proteins and RNA for the PTC in Thermus aquaticus.
Main Methods:
- Isolation and characterization of subribosomal particles from Thermus aquaticus large ribosomal subunits after extraction.
- Analysis of RNA and protein composition of active particles.
- N-terminal sequencing of identified ribosomal proteins.
- RNase treatment to assess protein accessibility.
Main Results:
- Active subribosomal particles contained 23S and 5S rRNA with eight specific ribosomal proteins (L2, L3, L13, L15, L17, L18, L21, L22).
- Ribosomal protein L4 was notably absent.
- Complete protein removal inactivated peptidyl transferase activity and disrupted tertiary folding.
- Protein accessibility increased after RNase treatment, suggesting an RNA cage structure.
Conclusions:
- The essential components for Thermus aquaticus peptidyl transferase activity are 23S rRNA and ribosomal proteins L2 and L3.
- The 50S ribosomal subunit may have a structural organization with an RNA cage surrounding core proteins.
- This finding refines our understanding of the minimal functional unit of the ribosome.