Global analysis of Escherichia coli RNA degradosome function using DNA microarrays

Abstract

RNase E, an essential endoribonuclease of Escherichia coli, interacts through its C-terminal region with multiple other proteins to form a complex termed the RNA degradosome. To investigate the degradosome's proposed role as an RNA decay machine, we used DNA microarrays to globally assess alterations in the steady-state abundance and decay of 4,289 E. coli mRNAs at single-gene resolution in bacteria carrying mutations in the degradosome constituents RNase E, polynucleotide phosphorylase, RhlB helicase, and enolase. Our results show that the functions of all four of these proteins are necessary for normal mRNA turnover. We identified specific transcripts and functionally distinguishable transcript classes whose half-life and abundance were affected congruently by multiple degradosome proteins, affected differentially by mutations in degradosome constituents, or not detectably altered by degradosome mutations. Our results, which argue that decay of some E. coli mRNAs in vivo depends on the action of assembled degradosomes, whereas others are acted on by degradosome proteins functioning independently of the complex, imply the existence of structural features or biochemical factors that target specific classes of mRNAs for decay by degradosomes.RNase E ͉ rhlB helicase ͉ enolase ͉ polynucleotide phosphorylase T he turnover of mRNA is a necessary component of normal genetic regulation within cells. In eubacteria, mRNA degradation results from the combined action of endo-and exoribonucleases. In Escherichia coli, the exoribonuclease polynucleotide phosphorylase (PNPase), the RhlB RNA helicase, and the glycolytic enzyme enolase assemble on the C-terminal region of the endoribonuclease RNase E as constituents of a protein complex termed the RNA degradosome (refs. 1-4; for recent reviews, see refs. 5 and 6). Two heat shock proteins, GroEL and DnaK (4), and polyphosphate kinase (Ppk) (7) also are associated with degradosomes in substoichiometric amounts. The N-terminal half of RNase E, which contains the catalytic domain of the enzyme (8), is not sufficient for degradosome formation (3, 9) but can associate the degradosome protein complex with the cytoplasmic membrane (11).The E. coli ams͞rne locus, which encodes RNase E, is required for bulk RNA turnover (12, 13) as well as for the processing of 9S rRNA (14,15). Investigations of the decay of individual transcripts (16)(17)(18)(19) and global investigations of mRNA abundance in rne mutants (20) have indicated a broadly important role for this enzyme. However, the RNase E region that provides a scaffold for degradosome formation is not essential for cell survival and growth (17,21,22), and truncated RNase E proteins lacking this domain are active in vivo as well as in vitro (8,23).Although the degradosome commonly has been viewed as an RNA decay ''machine'' (e.g., ref. 24), until recently (11) it was not known whether assembled degradosomes actually exist as such in living cells. Moreover, whether degradosome formation is a significant factor in mRNA decay in vivo has been...

RNaseE and the other constituents of the RNA degradosome are components of the bacterial cytoskeleton

“…The RNA degradosome is required for the normal maturation of transfer and ribosomal RNA and for degradation of most messenger RNAs (16)(17)(18). In degradosome-dependent mRNA decay, RhlB facilitates the degradation of structured RNA, and RNaseE provides the endoribonuclease activity that cuts the RNA into fragments that are further degraded by the 3Ј35Ј exoribonuclease activity of PNPase (reviewed in ref.…”

Structure of Escherichia coli RNase E catalytic domain and implications for RNA turnover

“…Such coordination may be partly achieved at the level of messenger RNA stability 1 , in which the targeted destruction of subsets of transcripts generates the potential for cross-regulating metabolic pathways. In Escherichia coli, the balance and composition of the transcript population is affected by RNase E, an essential endoribonuclease that not only turns over RNA but also processes certain key RNA precursors [2][3][4][5][6][7][8][9][10] . RNase E cleaves RNA internally, but its catalytic power is determined by the 5 0 terminus of the substrate, even if this lies at a distance from the cutting site [11][12][13][14] .…”

mRNA degradation and maturation in prokaryotes: the global players

“…The CTH of RNase E is not essential for catalytic activity and its removal has only a moderate effect on global mRNA decay (96,97). However, the CTH is important for the rapid degradation of many untranslated mRNA (98) and a microarray study shows that the assembled degradosome regulates the abundance of certain metabolic pathways in E. coli (99). The composition of the degradosome can be modified depending on conditions of growth or stress (100)(101)(102)(103).…”