Date of Award
1994
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Abstract
The genes of the Escherichia coli unc operon are differentially expressed according to the subunit requirements in the mature {dollar}\rm F\sb1F\sb0{dollar}-ATPase complex. The experiments described in this thesis were designed to elucidate how mRNA processing, degradation, and structure regulate the differential expression of the unc genes. Studies were focused on the uncBE region, near the 5{dollar}\sp\prime{dollar} end of the transcript, and uncDC region at the 3{dollar}\sp\prime{dollar} end of the transcript.;Northern blot analysis of uncC mRNA, the last gene of the operon, suggested that the unc transcription terminator protected the uncC message against exonucleolytic degradation, and that this stabilization was important in adequate synthesis of its gene product, the {dollar}\epsilon{dollar} subunit.;Northern blot and primer extension analysis of plasmid-encoded uncDC mRNA identified an RNase E-dependent endonucleolytic cleavage site 11 bases into the uncC coding region. This cleavage led to functional inactivation of the uncC mRNA and the upstream uncD mRNA, and consequently does not contribute to the differential relative expression of these genes.;A potential stem-loop structure 16 bases downstream of the uncC initiation codon, and 6 bases downstream of the aforementioned RNase E site, was subjected to site-directed mutagenesis to explore its significance in the RNase E-dependent cleavage, and in regulating the uncC expression. Results indicated that the structure was indispensable to the cleavage by RNase E, while the bases around the actual site were not so crucial. The expression studies uncovered a critical dependence of the stem-loop position in modulating the expression of uncC. The uncC expression was enhanced when the stem-loop was shifted downstream from the uncC ribosome binding site while moving it closer resulted in reduced expression of uncC. The results imply that the stem-loop provides steric hindrance to the binding of the ribosome and hence limits uncC expression.;Northern blot analysis of chromosomally-encoded unc mRNA indicated that its 5{dollar}\sp\prime{dollar} region also underwent RNase E-dependent processing. Northern blots of mRNA expressed from a plasmid carrying the uncBE genes from the 5{dollar}\sp\prime{dollar} region of the operon revealed that the uncB message was rapidly degraded by multiple internal cleavages, some dependent on functional RNase E. Primer extension analysis showed that the cleavages were made either in the uncB coding region or in the intercistronic region between uncB and uncE, the latter being the most 3{dollar}\sp\prime{dollar} cleavage. The rapid degradation of uncB message through cleavages at these sites provides a mechanism for segmental decay of unc mRNA to contribute to the differential expression of the unc genes.
Recommended Citation
Patel, Ashok M., "The Roles Ofmrna Stability And Secondary Structure In The Differential Expression Of The Escherichia Coliunc Genes" (1994). Digitized Theses. 2444.
https://ir.lib.uwo.ca/digitizedtheses/2444