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...

Whereas ribosomal proteins (r-proteins) are known primarily as components of the translational machinery, certain of these r-proteins have been found to also have extraribosomal functions. Here we report the novel ability of an r-protein, L4, to regulate RNA degradation in Escherichia coli. We show by affinity purification, immunoprecipitation analysis, and E. coli two-hybrid screening that L4 interacts with a site outside of the catalytic domain of RNase E to regulate the endoribonucleolytic functions of the enzyme, thus inhibiting RNase E-specific cleavage in vitro, stabilizing mRNAs targeted by RNase E in vivo, and controlling plasmid DNA replication by stabilizing an antisense regulatory RNA normally attacked by RNase E. Broader effects of the L4-RNase E interaction on E. coli transcripts were shown by DNA microarray analysis, which revealed changes in the abundance of 65 mRNAs encoding the stress response proteins HslO, Lon, CstA, YjiY, and YaeL, as well as proteins involved in carbohydrate and amino acid metabolism and transport, transcription/translation, and DNA/ RNA synthesis. Analysis of mRNA stability showed that the half lives of stress-responsive transcripts were increased by ectopic expression of L4, which normally increases along with other r-proteins in E. coli under stress conditions, and also by inactivation of RNase E. Our finding that L4 can inhibit RNase E-dependent decay may account at least in part for the elevated production of stress-induced proteins during bacterial adaptation to adverse environments.posttranscriptional control ͉ RNA degradation ͉ stress responses ͉ degradosome O ver the past two decades, an understanding of mRNA decay pathways in Escherichia coli has advanced significantly (for reviews, see ref. 1-3), and RNase E has emerged as a key player in mRNA turnover as well as in the processing and decay of noncoding RNAs (e.g., rRNAs [4,5], tRNAs [6,7], M1 RNA [8], and 6S RNA [9]). RNase E is a multifunctional endoribonuclease (10) known to preferentially cleave RNA within AU-rich singlestranded regions (11, 12) enriched in specific sequence determinants (13). The level of this enzyme in vivo is controlled via autoregulation of its own synthesis (14-16).In addition to its N-terminal catalytic domain (N-RNase E), RNase E contains a C-terminal region (C-RNase E) that serves as a scaffold (17, 18) for association with polynucleotide phosphorylase (PNPase), RhlB RNA helicase, and the glycolytic enzyme enolase to form the RNA-degrading complex known as the ''degradosome' ' (19, 20). C-terminal truncation of RNase E, which prevents degradosome assembly, leads to accumulation of RNase E-targeted mRNAs (21, 22), suggesting that degradosome assembly and functional interactions of degradosome components are necessary for normal mRNA turnover in E. coli.Although ribosomal proteins (r-proteins) function primarily as components of the translation machinery, some prokaryotic and eukaryotic r-proteins also have extraribosomal functions (23). For example, L4, an essential r-protein encoded by...

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 (␣ 3␤2) containing trimers of a catalytic subunit (␣) and dimers of another subunit (␤). The ␤-subunit has been believed to be enolase; we report here that it is instead the RhlB helicase. Whereas interaction between PNPase-␣ and enolase was observed in bacteria that synthesize RNase E having a scaffold region, immunoprecipitates from cells expressing PNPase-␣, RhlB, and enolase from single-copy chromosomal loci, plus a mutant RNase E protein lacking its C-terminal half, showed direct association of PNPase-␣ only with RhlB. Using affinity chromatography, we found that PNPase-␣ and RhlB form a ribonucleolytically active complex corresponding to the mass calculated previously for ␣3␤2 (i.e., 377-380 kDa), whereas no association between PNPase-␣ 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-␣ and RhlB occurs physiologically in the absence of the RNase E C-terminal region, that enolase association with PNPase-␣ is a consequence of the interaction of both proteins with RNase E, and that, contrary to current notions, enolase is not the ␤-subunit of E. coli PNPase complex.degradosome ͉ RNase E P olynucleotide phosphorylase (PNPase) is a major 3Ј to 5Ј exoribonuclease of Escherichia coli and functions both in the degradation of mRNA and stable RNA species and as a poly(A) polymerase (1, 2). The enzyme initially was discovered by Grunberg-Manago et al. in 1955 (3) in Azotobacter vinelandii. Subsequently, its enzymatic activity was detected among eubacteria (4, 5), Archea (6), eukaryotic microbes (7), plants (8), and animal cells (9). ''Degradosome'' complexes containing PNPase, RNase E, the RhlB helicase, enolase, and other proteins have been isolated from E. coli and other bacteria (10-13) and recently have been shown to exist also in vivo (14) and to function as ribonucleolytic machines (15,16). In yeast and animal cells, several PNPase-related 3Ј to 5Ј exonucleases have been identified and shown, along with an RNA helicase, to form an ''exosome '' complex (17-19). Because of its occurrence in a broad spectrum of organisms and its involvement in a variety of ribonucleolytic complexes (for recent reviews, see refs. 20 and 21), the identification and characterization of protein complexes containing PNPase continue to be of general interest.In crude cell extracts of E. coli, PNPase displays heterogeneity (22). Early in its history, PNPase was shown by Portier (23) to exist in two active forms, A or B, having molecular masses of Ϸ252 and 365 kDa, respective...

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