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Two modes of amber codon read-through in vitro.
Archives of Biochemistry and Biophysics
|May 1, 1985
Summary
Bacteriophage R17 coat protein synthesis in vitro is mainly due to reinitiation, not read-through. Amino acid misinsertion becomes dominant only at high Mg2+ concentrations.
Area of Science:
- Molecular Biology
- Bacteriophage Genetics
- Protein Synthesis
Background:
- Bacteriophage R17 coat cistron contains an amber mutation at the seventh codon.
- Read-through translation is generally inhibited by termination factors and ribosome-releasing factor (RRF).
Purpose of the Study:
- To investigate the mechanism of coat-like protein synthesis from bacteriophage R17 amB2 coat cistron in vitro.
- To reexamine the role of amino acid misinsertion versus reinitiation in producing coat-like protein.
Main Methods:
- In vitro translation using S30 extract from Escherichia coli nonsuppressor strain.
- Peptide fingerprinting and amino acid sequencing for protein characterization.
Main Results:
- A low level of coat-like protein synthesis was observed despite the presence of translation inhibitors.
- Peptide analysis revealed that the major population of coat-like protein arises from reinitiation at the eighth codon.
- Amino acid misinsertion at the amber codon becomes predominant only at Mg2+ concentrations above 16 mM.
Conclusions:
- Reinitiation of translation is the primary mechanism for coat-like protein production from bacteriophage R17 amB2 in this system.
- The contribution of amino acid misinsertion to read-through translation is dependent on Mg2+ concentration.