A <i>Streptomyces coelicolor</i> functional orthologue of <i>Escherichia coli</i> RNase E shows shuffling of catalytic and PNPase‐binding domains

Lee, Kangseok; Cohen, Stanley N.

doi:10.1046/j.1365-2958.2003.03435.x

Cited by 99 publications

(128 citation statements)

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“…Although the general nature of the conclusions have been found to be true in most bacteria, the exploration of other bacterial systems is providing us with novel information (6,8,30). In the present study we specifically examined the importance of RNase R, which is unique to the P. syringae degradosome.…”

Section: Discussionmentioning

confidence: 96%

Exoribonuclease R in Pseudomonas syringae Is Essential for Growth at Low Temperature and Plays a Novel Role in the 3′ End Processing of 16 and 5 S Ribosomal RNA

Rajyaguru

Madhuri

Ray

2007

Journal of Biological Chemistry

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The (3 3 5) exoribonuclease RNase R interacts with the endoribonuclease RNase E in the degradosome of the coldadapted bacterium Pseudomonas syringae Lz4W. We now present evidence that the RNase R is essential for growth of the organism at low temperature (4°C). Mutants of P. syringae with inactivated rnr gene (encoding RNase R) are cold-sensitive and die upon incubation at 4°C, a phenotype that can be complemented by expressing RNase R in trans. Overexpressing polyribonucleotide phosphorylase in the rnr mutant does not rescue the cold sensitivity. This is different from the situation in Escherichia coli, where rnr mutants show normal growth, but pnp (encoding polyribonucleotide phosphorylase) and rnr double mutants are nonviable. Interestingly, RNase R is not cold-inducible in P. syringae. Remarkably, however, rnr mutants of P. syringae at low temperature (4°C) accumulate 16 and 5 S ribosomal RNA (rRNA) that contain untrimmed extra ribonucleotide residues at the 3 ends. This suggests a novel role for RNase R in the rRNA 3 end processing. Unprocessed 16 S rRNA accumulates in the polysome population, which correlates with the inefficient protein synthesis ability of mutant. An additional role of RNase R in the turnover of transfer-messenger RNA was identified from our observation that the rnr mutant accumulates transfer-messenger RNA fragments in the bacterium at 4°C. Taken together our results establish that the processive RNase R is crucial for RNA metabolism at low temperature in the coldadapted Antarctic P. syringae.Regulated degradation of RNA within cells is mostly an outcome of coordinated and combined activities of endoribonucleases, exoribonucleases, and RNA helicases. In bacteria, few of these enzymes interact with each other to form the RNA degradosome complex (1-3). The degradosome has been shown to be involved in both mRNA and rRNA degradation (4, 5). In the Escherichia coli degradosome, RNase E (a 5Ј end-dependent endoribonuclease) associates with polynucleotide phosphorylase (PNPase, 2 a 3Ј 3 5Ј exoribonuclease), RhlB (a DEAD box RNA helicase), and enolase (an enzyme of glycolytic pathway) to constitute the "core" complex. Additionally, DnaK, poly(A) polymerase and polyphosphate kinase have also been reported to be a part of this complex (6). A similar kind of RNA degrading complex has also been reported from Rhodobacter capsulatus (7). On the other hand, we have recently shown that exoribonuclease RNase R interacts with RNase E in the degradosome of the cold-adapted Antarctic bacterium Pseudomonas syringae Lz4W (8). This bacterium does have a pnp gene that is expressed but does not form a part of this complex.3 PNPase and RNase R differ in their mode of action, the former exhibiting phosphorolytic activity and the latter exhibiting hydrolytic activity.RNase R is one of the eight exoribonucleases reported in E. coli and distributed widely in different prokaryotes (9). Although all of these exoribonucleases display 3Ј 3 5Ј activity on RNA substrate, RNase R is the most highly processive enzyme among ...

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Section: Discussionmentioning

confidence: 96%

Exoribonuclease R in Pseudomonas syringae Is Essential for Growth at Low Temperature and Plays a Novel Role in the 3′ End Processing of 16 and 5 S Ribosomal RNA

Rajyaguru

Madhuri

Ray

2007

Journal of Biological Chemistry

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“…So far, only the phosphate-dependent exonuclease PNPase was shown to be part of the E. coli degradosome (1), to interact with RNase E of S. coelicolor (12), or to co-purify with the R. capsulatus degradosome in substoichiometric amounts (13). Eukaryotic exosomes were also found to contain PNPase-like proteins (14,15), indicating the evolutionary conservation of phosphorolytic activities in the two protein complexes.…”

Section: Discussionmentioning

confidence: 99%

“…Not all bacteria have a gene for RNase E (rne), and some bacteria possess only short RNase E homologues without potential protein binding domains (12). They most probably do not harbor degradosomes.…”

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confidence: 99%

“…A second DEAD-box helicase also co-purifies with R. capsulatus RNase E. PNPase is not tightly associated with the complex but is found in substoichiometric amounts in RNase E-containing fractions after three purification steps (13). Recently, it was shown that a structurally shuffled RNase E homologue in Streptomyces coelicolor harbors its active center in the middle of the molecule and interacts with PNPase via its N-terminal part (12). The available data imply that the bacterial degradosome consists of RNase E, DEAD-box helicase(s), the phosphate-dependent exoribonuclease PNPase, and additional subunits, which vary in the different species.…”

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confidence: 99%

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Exoribonuclease R Interacts with Endoribonuclease E and an RNA Helicase in the Psychrotrophic Bacterium Pseudomonas syringae Lz4W

Rajyaguru

Klein

Sulthana

et al. 2005

Journal of Biological Chemistry

123

112

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Endoribonuclease E, a key enzyme involved in RNA decay and processing in bacteria, organizes a protein complex called degradosome. In Escherichia coli, Rhodobacter capsulatus, and Streptomyces coelicolor, RNase E interacts with the phosphate-dependent exoribonuclease polynucleotide phosphorylase, DEAD-box helicase(s), and additional factors in an RNA-degrading complex. To characterize the degradosome of the psychrotrophic bacterium Pseudomonas syringae Lz4W, RNase E was enriched by cation exchange chromatography and fractionation in a glycerol density gradient. Most surprisingly, the hydrolytic exoribonuclease RNase R was found to co-purify with RNase E. Co-immunoprecipitation and Ni 2؉ -affinity pull-down experiments confirmed the specific interaction between RNase R and RNase E. Additionally, the DEAD-box helicase RhlE was identified as part of this protein complex. Fractions comprising the three proteins showed RNase E and RNase R activity and efficiently degraded a synthetic stem-loop containing RNA in the presence of ATP. The unexpected association of RNase R with RNase E and RhlE in an RNA-degrading complex indicates that the cold-adapted P. syringae has a degradosome of novel structure. The identification of RNase R instead of polynucleotide phosphorylase in this complex underlines the importance of the interaction between endo-and exoribonucleases for the bacterial RNA metabolism. The physical association of RNase E with an exoribonuclease and an RNA helicase apparently is a common theme in the composition of bacterial RNAdegrading complexes.

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“…Thus, the major 39-59-exoribonuclease in S. coelicolor is almost certainly PNPase. Lee & Cohen (2001) have provided data suggesting that in S. coelicolor, PNPase and RNase E may be organized into a supramolecular complex like the E. coli degradosome.…”

Section: Discussionmentioning

confidence: 99%

RNA 3′-tail synthesis in Streptomyces: in vitro and in vivo activities of RNase PH, the SCO3896 gene product and polynucleotide phosphorylase

et al. 2006

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As in other bacteria, 39-tails are added post-transcriptionally to Streptomyces coelicolor RNA. These tails are heteropolymeric, and although there are several candidates, the enzyme responsible for their synthesis has not been definitively identified. This paper reports on three candidates for this role. First, it is confirmed that the product of S. coelicolor gene SCO3896, although it bears significant sequence similarity to Escherichia coli poly(A) polymerase I, is a tRNA nucleotidyltransferase, not a poly(A) polymerase. It is further shown that SCO2904 encodes an RNase PH homologue that possesses the polymerization and phosphorolysis activities expected for enzymes of that family. S. coelicolor RNase PH can add poly(A) tails to a model RNA transcript in vitro. However, disruption of the RNase PH gene has no effect on RNA 39-tail length or composition in S. coelicolor; thus, RNase PH does not function as the RNA 39-polyribonucleotide polymerase [poly(A) polymerase] in that organism. These results strongly suggest that the enzyme responsible for RNA 39-tail synthesis in S. coelicolor and other streptomycetes is polynucleotide phosphorylase (PNPase). Moreover, this study shows that both PNPase and the product of SCO3896 are essential. It is possible that the dual functions of PNPase in the synthesis and degradation of RNA 39-tails make it indispensable in Streptomyces.

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A Streptomyces coelicolor functional orthologue of Escherichia coli RNase E shows shuffling of catalytic and PNPase‐binding domains

Cited by 99 publications

References 58 publications

Exoribonuclease R in Pseudomonas syringae Is Essential for Growth at Low Temperature and Plays a Novel Role in the 3′ End Processing of 16 and 5 S Ribosomal RNA

Exoribonuclease R in Pseudomonas syringae Is Essential for Growth at Low Temperature and Plays a Novel Role in the 3′ End Processing of 16 and 5 S Ribosomal RNA

Exoribonuclease R Interacts with Endoribonuclease E and an RNA Helicase in the Psychrotrophic Bacterium Pseudomonas syringae Lz4W

RNA 3′-tail synthesis in Streptomyces: in vitro and in vivo activities of RNase PH, the SCO3896 gene product and polynucleotide phosphorylase

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