Global analysis of messenger RNA decay in Escherichia coli using DNA microarrays

The turnover of mRNA is necessary for the appropriate expression of a cell's genetic material. The adaptation of DNA microarrays for the experimental determination of mRNA half-lives as presented in this dissertation allowed for examination of this process in E. coli at single gene resolution on a genomic scale. In studies of the wild-type strain MG1655 we found that numerous transcript primary and secondary structural features that affect the decay of individual mRNAs are not predictive of message stability on a genome wide basis. However, we observed that the function of a transcript's encoded protein was associated with its stability. By experimentally determining the relative abundance of individual transcripts and then comparing the resulting data to measured mRNA stabilities, we found that while certain abundant transcripts have longer than average stability, greater abundance is more often associated with rapid mRNA turnover. Our observations support the possibility that E. coli rapidly turns over abundant transcripts encoding proteins in specific functional categories in order to allow their rapid down regulation in response to external stimuli or internal events such as cell cycle progression.;Transcript abundance profiling in mutant cells allowed us to identify probable mRNA targets for several proteins believed to be involved in messenger turnover. In particular, we identified transcripts affected by ribonucleases (RNases) E and G. Other studies showed that the ability of RNase G to complement cells lacking RNase E is associated with the restoration of only 105 transcripts, predominantly involved in energy generation, to more normal levels. Studies of transcript levels in additional mutants enabled us to define sets of transcripts that displayed common alterations in abundance in response to mutations in multiple degradosome component proteins. The finding that transcript abundance can be congruently affected by mutations in RNase E, polynucleotide phosphorylase (PNPase), RhlB helicase and enolase provided evidence that the complex functions as an assembled unit in vivo. Finally, determinations of mRNA half-lives in cells lacking the function of each of these core degradosome components indicate that the RhlB helicase and enolase as well as RNase E and PNPase are necessary for normal mRNA decay.