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Escherichia coli polynucleotide phosphorylase (PNPase), a protein that has both ribonucleolytic and synthetic capabilities, binds, along with the 48-kDa glycolytic enzyme enolase, the 50-kDa DEAD-box protein RhlB helicase and other cellular proteins to the C-terminal 'scaffold' region of RNase E to form a complex termed the RNA degradosome. PNPase itself has been reported to exist as a complex ([[alpha].sub.3][[beta].sub.2]) containing trimers of a catalytic subunit ([alpha]) and dimers of another subunit ([beta]). The [beta]-subunit has been believed to be enolase; we report here that it is instead the RhlB helicase. Whereas interaction between PNPase-[alpha] and enolase was observed in bacteria that synthesize RNase E having a scaffold region, immunoprecipitates from cells expressing PNPase-[alpha], RhlB, and enolase from single-copy chromosomal loci, plus a mutant RNase E protein lacking its C-terminal half, showed direct association of PNPase-[alpha] only with RhIB. Using affinity chromatography, we found that PNPase-[alpha] and RhIB form a ribonucleolytically active complex corresponding to the mass calculated previously for [[alpha].sub.3][[beta.sub.2] (i.e., 377-380 kDa), whereas no association between PNPase-[alpha] and enolase was detected. Chromosomal deletion of the eno gene had no effect on the ability of PNPase to degrade either single- or double-stranded RNAs. Collectively, our findings show that direct interaction between PNPase-[alpha] and RhIB occurs physiologically in the absence of the RNase E C-terminal region, that enolase association with PNPase-[alpha] is a consequence of the interaction of both proteins with RNase E, and that, contrary to current notions, enolase is not the [beta]-subunit of E. coli PNPase complex. degradosome | RNase E

The decay of mRNA plays an important role in the regulation of gene expression. Although relatively ignored for many years and regarded as a simple ribonucleotide salvage pathway, mRNA decay has been established in recent years as a well-defined cellular process that plays an integral role in determining gene expression. The recent application of microarray methods to the study of diverse organisms will help us to better understand these gene regulatory circuits and the influence of transcript stability on gene expression. DNA microarray technology is the method of choice to study individual mRNA half-lives on a global scale. It is important to standardize these methods to generate reproducible and reliable results. In this chapter, we describe experimental designs for the analysis of mRNA decay on a genome-wide scale and provide detailed protocols for each experimental step. We also present an analysis of the decay of chromosomally encoded mRNAs in E. coli.