Organization and Expression of the Polynucleotide Phosphorylase Gene (
 pnp
 ) of
 Streptomyces
 : Processing of
 pnp
 Transcripts in
 Streptomyces antibioticus

Bralley, Patricia; Jones, George H.

doi:10.1128/jb.186.10.3160-3172.2004

Cited by 20 publications

(32 citation statements)

References 54 publications

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“…To our knowledge, no such site has been demonstrated in E. coli. Although the start point for the pnp transcript has not been determined in S. coelicolor, studies from Streptomyces antibioticus suggest that the pnp transcript may not be cleaved by RNase III upstream of the coding region (2). If this is the case in S. coelicolor, the internal cleavage site explains the observation that the level of the pnp transcript, like that of the readthrough transcript, increases in the absence of RNase III (Fig.…”

Section: Discussionmentioning

confidence: 87%

“…In Streptomyces coelicolor and Streptomyces antibioticus, the phosphorolytic activity of PNPase is modulated by nucleoside diphosphates (11) and both phosphorolysis and polymerization are modulated by the alarmone, (p)ppGpp (13). As is the case in E. coli, the PNPase gene, pnp, is a part of an operon that includes the rpsO gene, which encodes ribosomal protein S15 (2). In E. coli, there is an intergenic hairpin, situated between rpsO and pnp, that is a site for processing by the double-strand-specific endonuclease, RNase III (17,29,30).…”

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confidence: 99%

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

2011

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We have examined the expression of the rpsO-pnp operon in an RNase III (rnc) mutant of Streptomyces coelicolor. Western blotting demonstrated that polynucleotide phosphorylase (PNPase) levels increased in the rnc mutant, JSE1880, compared with the parental strain, M145, and this observation was confirmed by polymerization assays. It was observed that rpsO-pnp mRNA levels increased in the rnc mutant by 1.6-to 4-fold compared with M145. This increase was observed in exponential, transition, and stationary phases, and the levels of the readthrough transcript, initiated upstream of rpsO in the rpsO-pnp operon; the pnp transcript, initiated in the rpsO-pnp intergenic region; and the rpsO transcript all increased. The increased levels of these transcripts in JSE1880 reflected increased chemical half-lives for each of the three. We demonstrated further that overexpression of the rpsO-pnp operon led to significantly higher levels of PNPase activity in JSE1880 compared to M145, reflecting the likelihood that PNPase expression is autoregulated in an RNase IIIdependent manner in S. coelicolor. To explore further the increase in the level of the pnp transcript initiated in the intergenic region in JSE1880, we utilized that transcript as a substrate in assays employing purified S. coelicolor RNase III. These assays revealed the presence of hitherto-undiscovered sites of RNase III cleavage of the pnp transcript. The position of those sites was determined by primer extension, and they were shown to be situated in the loops of a stem-loop structure.Polynucleotide phosphorylase (PNPase) is a 3Ј-5Ј-exoribonuclease that functions in the phosphorolytic degradation of RNA molecules in bacteria and in eukaryotic organelles (14,21). In Escherichia coli and other bacteria, PNPase plays an important role in the degradation of mRNAs. Thus, endonucleolytic cleavage of RNA molecules generates 3Ј ends that are substrates for the action of PNPase and RNase II, an exonuclease that functions hydrolytically (8,12,28). PNPase plays another role in E. coli, at least under some circumstances. As is the case in eukaryotes, the 3Ј ends of at least some RNA molecules in bacteria are polyadenylated (31, 32). Polyadenylation facilitates the degradation of RNAs in bacteria (22,24,27). While the major enzyme responsible for RNA polyadenylation in E. coli is poly(A) polymerase I (PAP I [7]), mutants of E. coli lacking PAP I still retain the ability to polyadenylate RNAs (26), indicating that there is at least one other polyadenylating enzyme in those cells. Mohanty and Kushner have presented evidence indicating that the second PAP in E. coli is none other than PNPase (25). They argue that under appropriate conditions in vivo, PNPase can serve to degrade RNAs or synthesize poly(A) tails and that this enzyme is responsible for the G, C, and U residues that are found at low frequency in the poly(A) tails of RNAs from wild-type E. coli (25).PNPase structure, function, and expression have been studied extensively in species of the soil-dwelling, antibiotic-pro...

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Section: Discussionmentioning

confidence: 87%

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confidence: 99%

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

2011

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“…RNA-Seq analysis of these preparations indicated that mRNA reads represented 64% of the total for M145 and and JSE1880 (rnc-null mutant). RT-PCR was performed as described previously (8,16), and products were analyzed after 20 PCR cycles to ensure linearity between band intensity and the number of cycles. Lane 1, size standards (the arrow indicates the 500-bp standard); lane 2, mRNA from M145 as the template for reverse transcription; lane 3, M145 BARD RNA; lane 4, M145 mock BARD RNA; lanes 5 and 8, JSE1880 mRNA; lanes 6 and 9, JSE1880 BARD RNA; lanes 7 and 10, JSE1880, mock BARD RNA.…”

Section: Resultsmentioning

confidence: 99%

“…Reverse transcription-PCRs (RT-PCRs) were performed using random primers for reverse transcription and gene-specific primers for PCR as described previously (8,16). The results shown in Fig.…”

Section: Methodsmentioning

confidence: 99%

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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“…This 333 base fragment was produced by RNase III cleavage of a transcript obtained from a cloned DNA fragment representing the rpsO-pnp intergenic region (Bralley & Jones, 2004). The transcript was synthesized using T7 RNA polymerase and a-[ 32 P]CTP, and the 39-end of the RNase III digestion product represents the primary RNase III processing site in the rpsO-pnp intergenic region.…”

Section: Methodsmentioning

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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Organization and Expression of the Polynucleotide Phosphorylase Gene ( pnp ) of Streptomyces : Processing of pnp Transcripts in Streptomyces antibioticus

Cited by 20 publications

References 54 publications

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

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

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

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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