RNase III-Dependent Expression of the
            <i>rpsO-pnp</i>
            Operon of Streptomyces coelicolor

Gatewood, Marcha L.; Bralley, Patricia; Jones, George H.

doi:10.1128/jb.00452-11

Cited by 15 publications

(23 citation statements)

References 37 publications

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“…Quantification of the band intensities indicated that the JSE1880 mRNA preparation (lane 5) contained ca. 2.5 times as much of the readthrough transcript as the M145 preparation (lane 2), a value consistent with the levels observed in Northern blots of RNAs from M145 and JSE1880 (15). Figure 1 also shows that the JSE1880 mRNA preparation contained transcripts of the ramR gene (lane 8), which encodes a response regulator (35).…”

Section: Resultssupporting

confidence: 62%

“…2 and compared those results with previous data obtained for these two transcripts by Northern blotting. Our Northern blotting studies indicated a 1.6-fold increase in the rpsO transcript level, encoding ribosomal protein S15, in the mRNA pools from JSE1880 compared to that of M145 during exponential growth (15). The corresponding ratio obtained from the RNA-Seq data was ca.…”

Section: Resultsmentioning

confidence: 75%

“…Thus, the rspO-pnp transcript was expected to be present in both M145 and JSE1880, as is shown in Fig. 1, with higher levels of this transcript present in the mutant strain lacking RNase III (15). Quantification of the band intensities indicated that the JSE1880 mRNA preparation (lane 5) contained ca.…”

Section: Resultsmentioning

confidence: 92%

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RNA-Seq and RNA Immunoprecipitation Analyses of the Transcriptome of Streptomyces coelicolor Identify Substrates for RNase III

et al. 2012

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RNase III is a key enzyme in the pathways of RNA degradation and processing in bacteria and has been suggested as a global regulator of antibiotic production in Streptomyces coelicolor. Using RNA-Seq, we have examined the transcriptomes of S. coelicolor M145 and an RNase III (rnc)-null mutant of that strain. RNA preparations with reduced levels of structural RNAs were prepared by subtractive hybridization prior to RNA-Seq analysis. We initially identified 7,800 transcripts of known and putative proteincoding genes in M145 and the null mutant, JSE1880, along with transcripts of 21 rRNA genes and 65 tRNA genes. Approximately 3,100 of the protein-coding transcripts were categorized as low-abundance transcripts. For further analysis, we selected those transcripts of known and putative protein-coding genes whose levels changed by >2-fold between the two S. coelicolor strains and organized those transcripts into 16 functional categories. We refined our analysis by performing RNA immunoprecipitation of the mRNA preparation from JSE1880 using a mutant RNase III protein that binds to transcripts but does not cleave them. This analysis identified ca. 800 transcripts that were enriched in the RNA immunoprecipitates, including 28 transcripts whose levels also changed by >2-fold in the RNA-Seq analysis. We compare our results with those obtained by microarray analysis of the S. coelicolor transcriptome and with studies describing the characterization of small noncoding RNAs. We have also used the RNA immunoprecipitation results to identify new substrates for RNase III cleavage.T he double-strand-specific endonuclease RNase III is found in bacteria and eukaryotes (12,25,33). In addition to its general role in RNA processing, RNase III is involved in the regulation of gene expression in Escherichia coli and other bacteria. Thus, RNase III autoregulates its own expression in E. coli and is also involved in the regulation of the expression of the polynucleotide phosphorylase (PNPase) gene, pnp (3, 40). There are a number of other examples of the regulation of gene expression by RNase III in E. coli and other bacteria (reviewed in references 12, 25, and 33).Streptomyces are Gram-positive soil bacteria notable for their ability to sporulate and for the production of antibiotics (6,7,11). Members of the genus Streptomyces produce nearly 70% of all antibiotics used in clinical and veterinary medicine worldwide (5). Much is known about the regulation of antibiotic production in the paradigm for the study of antibiotic production in streptomycetes, Streptomyces coelicolor (6, 7). Of particular relevance to the present report are the studies of Champness and coworkers (1, 2) on the absB locus of S. coelicolor. absB was shown to encode a homolog of E. coli RNase III, and the function of the absB product as a double-strand-specific endoribonuclease has been subsequently verified (1, 2, 10). Mutations in the absB locus reduce or abolish the production of all four of the antibiotics normally synthesized by S. coelicolor (2). Moreover, Acet...

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Section: Resultssupporting

confidence: 62%

Section: Resultsmentioning

confidence: 75%

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RNA-Seq and RNA Immunoprecipitation Analyses of the Transcriptome of Streptomyces coelicolor Identify Substrates for RNase III

et al. 2012

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“…Overexpression of PNPase led to decreased antibiotic production in Streptomyces antibioticus (155). Since the expression of pnp is RNase III dependent in S. coelicolor (156), it will be interesting to investigate the connection of PNPase, RNase III, and antibiotic production. Little is known about the functions of other RNases in Streptomyces.…”

Section: Rna Regulation Of Antibiotic Productionmentioning

confidence: 99%

Molecular Regulation of Antibiotic Biosynthesis in Streptomyces

Liu

Chater

Chandra

et al. 2013

Microbiol Mol Biol Rev

592

536

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SUMMARY Streptomycetes are the most abundant source of antibiotics. Typically, each species produces several antibiotics, with the profile being species specific. Streptomyces coelicolor , the model species, produces at least five different antibiotics. We review the regulation of antibiotic biosynthesis in S. coelicolor and other, nonmodel streptomycetes in the light of recent studies. The biosynthesis of each antibiotic is specified by a large gene cluster, usually including regulatory genes (cluster-situated regulators [CSRs]). These are the main point of connection with a plethora of generally conserved regulatory systems that monitor the organism's physiology, developmental state, population density, and environment to determine the onset and level of production of each antibiotic. Some CSRs may also be sensitive to the levels of different kinds of ligands, including products of the pathway itself, products of other antibiotic pathways in the same organism, and specialized regulatory small molecules such as gamma-butyrolactones. These interactions can result in self-reinforcing feed-forward circuitry and complex cross talk between pathways. The physiological signals and regulatory mechanisms may be of practical importance for the activation of the many cryptic secondary metabolic gene cluster pathways revealed by recent sequencing of numerous Streptomyces genomes.

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“…1B). This substrate, derived from the S. coelicolor rpsO-pnp operon, contains the rpsO terminator and the intergenic hairpin that is the substrate for RNase III (18). Figure 3A shows a typical polyacrylamide gel separation of the products of RP3 phosphorolysis using wild-type PNPase.…”

Section: Rationale For the Construction Of The Pnpase Mutantsmentioning

confidence: 99%

Streptomyces coelicolor Polynucleotide Phosphorylase Can Polymerize Nucleoside Diphosphates under Phosphorolysis Conditions, with Implications for the Degradation of Structured RNAs

Jones

Mackie

2013

J Bacteriol

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We have examined the ability of wild-type polynucleotide phosphorylase (PNPase) from Streptomyces coelicolor and two mutant forms of the enzyme, N459D and C468A, to function in the polymerization of ADP and in the phosphorolysis of RNA substrates derived from the S. coelicolor rpsO-pnp operon. The wild-type enzyme was twice as active in polymerization as N459D and four times as active as C468A. The k cat /K m value for phosphorolysis of a structured RNA substrate by N459D was essentially the same as that observed for the wild-type enzyme, while C468A was 50% as active with this substrate. A mixture of all four common nucleoside diphosphates increased the k cat /K m for phosphorolysis of the structured substrate by the wild-type enzyme by a factor of 1.7 but did not affect phosphorolysis catalyzed by N459D or C468A. We conducted phosphorolysis of the structured substrate in the presence of nucleoside diphosphates and labeled the 3= ends of the products of those reactions using [ 32 P]pCp. Digestion of the end-labeled RNAs and display of the products on a sequencing gel revealed that wild-type S. coelicolor PNPase was able to synthesize RNA 3= tails under phosphorolysis conditions while the N459D and C468A mutants could not. The wild-type enzyme did not add 3= tails to a substrate that already possessed an unstructured 3= tail. We propose a model in which the transient synthesis of 3= tails facilitates the phosphorolysis of structured substrates by Streptomyces PNPase.

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RNase III-Dependent Expression of the rpsO-pnp Operon of Streptomyces coelicolor

Cited by 15 publications

References 37 publications

RNA-Seq and RNA Immunoprecipitation Analyses of the Transcriptome of Streptomyces coelicolor Identify Substrates for RNase III

RNA-Seq and RNA Immunoprecipitation Analyses of the Transcriptome of Streptomyces coelicolor Identify Substrates for RNase III

Molecular Regulation of Antibiotic Biosynthesis in Streptomyces

Streptomyces coelicolor Polynucleotide Phosphorylase Can Polymerize Nucleoside Diphosphates under Phosphorolysis Conditions, with Implications for the Degradation of Structured RNAs

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