Related Experiment Videos
CCA addition by tRNA nucleotidyltransferase: polymerization without translocation?
P Y Shi1, N Maizels, A M Weiner
1Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06520-8024, USA.
The EMBO Journal
|June 26, 1998
Summary
CCA-adding enzymes use a fixed tRNA binding site to repair the 3' end of transfer RNA (tRNA). This mechanism ensures accurate nucleotide addition for essential protein synthesis.
Area of Science:
- Molecular Biology
- Biochemistry
- Enzymology
Background:
- Transfer RNAs (tRNAs) are crucial for protein synthesis, requiring a conserved 3'-terminal CCA sequence for function.
- CCA-adding enzymes are responsible for repairing and maintaining this essential 3'-CCA sequence on all tRNAs.
- Understanding tRNA recognition by CCA-adding enzymes is key to elucidating tRNA maturation and function.
Purpose of the Study:
- To investigate the critical contacts between tRNA substrates and both archaeal (Sulfolobus shibatae class I) and bacterial (Escherichia coli class II) CCA-adding enzymes.
- To determine how these enzymes recognize and bind tRNA during the CCA addition process.
- To elucidate the mechanism of CCA sequence repair and its regulation.
Main Methods:
- Chemical probing using alkylation of specific tRNA phosphates in the acceptor stem and TPsiC stem-loop.
- Assessing the impact of alkylation on CTP and ATP addition to tRNA substrates.
- Cross-linking of tRNA-C to the S. shibatae enzyme to evaluate residual activity.
- Comparative analysis of substrate binding and protection patterns between class I and class II enzymes.
Main Results:
- Alkylation of specific tRNA phosphates in the acceptor stem and TPsiC stem-loop significantly inhibited both CTP and ATP addition by both enzymes.
- Both archaeal and bacterial CCA-adding enzymes protected the same tRNA phosphates in tRNA-C and tRNA-CC substrates.
- tRNA-C cross-linked to the S. shibatae enzyme retained full activity for CTP and ATP addition, indicating a fixed substrate-enzyme interaction.
- These findings suggest a conserved mechanism of tRNA recognition involving a stable binding interface.
Conclusions:
- The tRNA substrate remains fixed on the enzyme surface throughout the CCA addition process.
- A single active site likely reuses a CTP binding site, with the ATP binding site formed dynamically by the refolded tRNA terminus and the enzyme.
- Nucleotide addition ceases when the binding pocket is optimally filled, ensuring accurate CCA sequence formation.
- The dynamic ribonucleoprotein structure serves as the template for CCA addition, highlighting a sophisticated enzymatic mechanism.