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Sequence changes preceding a Shine-Dalgarno region influence trpE mRNA translation and decay
1Department of Biological Sciences, Stanford University, CA 94305-5020.
Journal of Molecular Biology
|November 5, 1988
Summary
Mutations in the Escherichia coli trpE gene's pre-Shine-Dalgarno region significantly boosted trpE protein levels. These changes enhanced trpE mRNA stability and translation, improving enzyme activity.
Area of Science:
- Molecular Biology
- Microbial Genetics
Background:
- The Escherichia coli trpE promoter system was expected to yield higher trpE protein levels.
- Observed lower-than-expected trpE protein production necessitated further investigation.
Purpose of the Study:
- To investigate the impact of mutations in the pre-Shine-Dalgarno region on trpE expression.
- To analyze the effects on trpE mRNA synthesis, translation, and decay.
Main Methods:
- Introduction of deletion, insertion, and substitution mutations in the pre-Shine-Dalgarno region.
- Assay of trpE enzyme activity.
- Measurement of trpE mRNA levels and stability using pulse-chase analysis.
Main Results:
- Certain pre-Shine-Dalgarno mutations increased trpE enzyme activity by two- to fivefold.
- These mutations led to elevated steady-state levels of trpE mRNA.
- mRNA degradation studies indicated that increased trpE mRNA stability correlated with higher expression levels.
Conclusions:
- Mutational changes in the pre-Shine-Dalgarno region can significantly enhance trpE expression in E. coli.
- The primary mechanism for increased trpE expression appears to be enhanced trpE mRNA stability, potentially alongside translation effects.