The control of mRNA stability can be an important element in the post-transcriptional regulation of gene expression. Little is known about how the stability of different messages is determined. This application describes experiments which are designed to analyze the process of mRNA decay in two prokaryotic organisms, Bacillus subtilis and Escherichia coli. Our approach is to concentrate on the decay of RNA encoded by one gene, the ermC gene carried on plasmid pE194 in B. subtilis, which confers erythromycin resistance on the host. Translation of ermC mRNA is inducible by erythromycin, and under inducing conditions the stability of ermC mRNA increases to about 20 times the level of most bacterial mRNAs. The ability to manipulate ermC mRNA stability by the presence or absence of the inducer, in addition to the fact that ermC gene expression and mRNA structure are well-characterized, makes ermC a good system for studying mechanisms of mRNA decay. The specific ojectives of this proposal are the following: 1. Determine the mechanism of induced ermC mRNA stabilization by constructing insertion and deletion mutations in the portion of ermC responsible for induced stability (the promoter-proximal region). The decay of ermC mRNA encoded by these mutants is followed by isolating RNA at increasing times after transcription inhibition and probing for ermC by Northern blot hybridization. 2. Construct and analyze transcriptional fusions which contain the promoter-proximal region of ermC and parts of other, unstable mRNAs in order to study the cis-dominance of ermC sequences in conferring induced stability. 3. Isolate and characterize plasmid and host mutants which allow stability of uninduced ermC mRNA. A selection scheme for isolating such mutants in E. coli is presented. 4. Establish an assay for host factors involved in mRNA decay by the use of a B. Subtilis in vitro transcription/translation system in which ermC mRNA appears to be stable, even under non-inducing conditions.
|Effective start/end date||1/02/88 → 31/01/93|
- National Institute of General Medical Sciences
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