Related Experiment Videos
Codon usage and mistranslation. In vivo basal level misreading of the MS2 coat protein message
Abstract:
The coat protein of the small RNA virus MS2 shows charge heterogeneity in vivo. In most strains there is a basic satellite of the native protein. We have shown that this basic satellite is greatly diminished or absent in strains with the streptomycin-resistant allele, rpsL, a mutation which leads to increased translational accuracy. Further, the satellite is present in cells where the coat protein is encoded by duplex DNA. Tryptic digests of the satellite show that it contains new lysine-containing peptides which appear to be the same as those found in derivatives of coat protein which have a lysine for asparagine substitution. Sequencing of the NH2-terminal 19 amino acids of the satellite protein shows that the asparagine codon AAU at amino acid 12 is misread approximately 8 times more frequently than the AAC at amino acid 3. We conclude that the satellite species is the result of basal level lysine for asparagine substitution. These substitutions are most likely caused by preferential misreading of AAU codons at a frequency of approximately 5 X 10(-3), 10-fold higher than the average error frequency.
Insights
The MS2 virus coat protein exhibits charge variations due to lysine substitutions for asparagine. This phenomenon is linked to translational accuracy and codon misreading, impacting protein function.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- The MS2 virus coat protein displays charge heterogeneity in vivo.
- A basic satellite protein is typically observed in most MS2 strains.
Purpose of the Study:
- To investigate the cause of the basic satellite in MS2 coat protein.
- To determine the relationship between translational accuracy and satellite formation.
Main Methods:
- Analysis of MS2 strains with streptomycin-resistant allele (rpsL).
- Comparison of coat protein from DNA-encoded vs. RNA-encoded sources.
- Tryptic digestion and amino acid sequencing of the satellite protein.
Main Results:
- The basic satellite was diminished in strains with the rpsL allele, indicating increased translational accuracy.
- Satellite protein contained new lysine-containing peptides, suggesting amino acid substitutions.
- Sequencing revealed frequent misreading of asparagine codon AAU at position 12, leading to lysine substitution.
Conclusions:
- The basic satellite is formed by basal-level lysine for asparagine substitutions.
- These substitutions result from preferential misreading of AAU codons at a frequency of approximately 5 x 10(-3).
- This misreading is approximately 10-fold higher than the average error frequency, impacting MS2 coat protein integrity.