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

Bralley, Patricia; Gust, Bertolt; Chang, Sheng; Chater, Keith F.; Jones, George H.

doi:10.1099/mic.0.28363-0

Cited by 30 publications

(34 citation statements)

References 46 publications

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“…This structure could play a role in transcription termination or transcript stability, and its exclusion from the duplicated region in strain FL2435 could explain why the yield improvement effect was not seen in this strain (Pulido and Jimenez, 1987). Other mechanisms that could affect regulation of the MCM operon or stability of its mRNA transcript (Bralley et al, 2006) could be involved and certainly more experimentation will be required to clarify the role of mutR or other factors on the strain improvement effect.…”

Section: Discussionmentioning

confidence: 99%

Engineering of the methylmalonyl-CoA metabolite node of Saccharopolyspora erythraea for increased erythromycin production

Reeves

Brikun

Cernota

et al. 2007

Metabolic Engineering

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Engineering of the methylmalonyl-CoA (mmCoA) metabolite node of the Saccharopolyspora erythraea wild type strain (FL2267) through duplication of the mmCoA mutase (MCM) operon led to a 51% (range 40%-64%, 0.95 CI, N = 152) increase in erythromycin production in a highperformance oil-based fermentation medium. The MCM operon was carried on a 6.8 kb DNA fragment in plasmid pFL2212 which was inserted by homologous recombination into the S. erythraea chromosome. The fragment contained one uncharacterized gene, ORF1; three MCM related genes, mutA, mutB, meaB; and one gntR-family regulatory gene, mutR. Additional strains were constructed containing partial duplications of the MCM operon, as well as a knockout of ORF1, none of these strains showed any significant alteration in their erythromycin production profile. The combined results showed that increased erythromycin production only occurred in strain FL2385 containing a duplication of the entire MCM operon including mutR and a predicted stem-loop structure overlapping the 3′ terminus of the mutR coding sequence.

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Section: Discussionmentioning

confidence: 99%

Engineering of the methylmalonyl-CoA metabolite node of Saccharopolyspora erythraea for increased erythromycin production

Reeves

Brikun

Cernota

et al. 2007

Metabolic Engineering

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“…For the TNT reactions, an E. coli tRNA Asp lacking either the 3Ј-terminal CA residues or the terminal A residue and prepared by transcription of plasmid pmBsDCCA (15) as described in Materials and Methods was used as the substrate. NTSFII and NTSFIII were examined for C-and A-adding TNT activity, and the S. coelicolor SCO3896 gene product, which has been shown to be a TNT (2,21), was used as a positive control for C and A addition to tRNA 3Ј ends. Products from these reactions were separated by polyacrylamide gel electrophoresis on 5 or 10% denaturing gels, followed by autoradiography of the gels, and the results are shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…4, the 3Ј tails associated with the Geobacter RNAs are heteropolymeric, containing G, C, and U residues as well as A residues. In E. coli, Streptomyces, some cyanobacteria, and spinach chloroplasts, polynucleotide phosphorylase is the enzyme responsible for the addition of G, C, and U residues to RNA 3Ј tails (2,14,17,21,25). This may also be the case in G. sulfurreducens, which, like all other bacteria except mycoplasmas, contains a gene for polynucleotide phosphorylase.…”

Section: Vol 191 2009mentioning

confidence: 99%

“…Transcription of the BbsI-digested template produced tRNA Asp -CC, while transcription of the FokI-digested template produced tRNA Asp -C. Enzyme assays. Generally, PAP and TNT assays were performed using 60-l reaction mixtures as described previously (1,2,5 To examine the products of the TNT and PAP reactions, large-scale mixtures (60 to 240 l) were incubated as described above and extracted with phenol-chloroform. Labeled RNAs were precipitated with ethanol.…”

Section: Methodsmentioning

confidence: 99%

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Geobacter sulfurreducens Contains Separate C- and A-Adding tRNA Nucleotidyltransferases and a Poly(A) Polymerase

2009

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The genome of Geobacter sulfurreducens contains three genes whose sequences are quite similar to sequences encoding known members of an RNA nucleotidyltransferase superfamily that includes tRNA nucleotidyltransferases and poly(A) polymerases. Reverse transcription-PCR using G. sulfurreducens total RNA demonstrated that the genes encoding these three proteins are transcribed. These genes, encoding proteins designated NTSFI, NTSFII, and NTSFIII, were cloned and overexpressed in Escherichia coli. The corresponding enzymes were purified and assayed biochemically, resulting in identification of NTSFI as a poly(A) polymerase, NTSFII as a C-adding tRNA nucleotidyltransferase, and NTSFIII as an A-adding tRNA nucleotidyltransferase. Analysis of G. sulfurreducens rRNAs and mRNAs revealed the presence of heteropolymeric RNA 3 tails. This is the first characterization of a bacterial system that expresses separate C-and A-adding tRNA nucleotidyltransferases and a poly(A) polymerase.RNA nucleotidyltransferases play an important role in the maturation, processing, and degradation of RNAs. tRNA nucleotidyltransferases (TNTs) and poly(A) polymerases (PAPs) are both members of a superfamily of nucleotidyltransferases (13, 27). TNTs add C and A residues to the 3Ј ends of immature or damaged tRNA molecules and are found in bacteria, archaea, and eukaryotes (24). PAPs, which are present in both eukaryotes and bacteria, polyadenylate RNA 3Ј ends (27). With regard to the distribution of RNA nucleotidyltransferases in bacteria, with only a few exceptions, the genomes of the alpha-and epsilonproteobacteria and of the low-and high-GϩC-content gram-positive bacteria appear to encode only one member of this superfamily, presumably a CCA-adding TNT (1). Several of the so-called deeply branching bacterial species, including Aquifex aeolicus and Deinococcus radiodurans, and some cyanobacteria contain two RNA nucleotidyltransferases, which have been shown to function as separate C-and A-adding TNTs (22,23). Interestingly, at least one low-GϩC-content gram-positive species, Bacillus halodurans, has also been shown to contain separate C-and A-adding TNTs (1). The beta-and gammaproteobacteria, such as Escherichia coli, also contain two RNA nucleotidyltransferases, a CCA-adding TNT and a PAP, PAP I, that adds 3Ј tails to the ends of RNA molecules in cells of these organisms (8,11,18,19). mRNAs and 16S and 23S rRNAs are polyadenylated in E. coli and presumably in other beta-and gammaproteobacteria, and polyadenylation facilitates the exonucleolytic degradation of these RNAs by RNase II and polynucleotide phosphorylase (9,16,18,19).The genomes of several deltaproteobacteria also encode two members of the RNA nucleotidyltransferase superfamily (NTSF), and the genomes of a few deltaproteobacteria appear to encode three NTSF enzymes (1). In particular, the genomes of Geobacter sulfurreducens, Geobacter metallireducens, Pelobacter carbinolicus, and Desulfotalea psychrophila all contain three open reading frames with significant homology to open reading ...

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“…Streptomyces members do not appear to contain PAP I, and the enzyme responsible for the synthesis of RNA 3= tails in members of that genus is thought to be PNPase (9,10). In Streptomyces coelicolor and Streptomyces antibioticus, the phosphorolytic activity of PNPase is modulated by nucleoside diphosphates.…”

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

Cited by 30 publications

References 46 publications

Engineering of the methylmalonyl-CoA metabolite node of Saccharopolyspora erythraea for increased erythromycin production

Engineering of the methylmalonyl-CoA metabolite node of Saccharopolyspora erythraea for increased erythromycin production

Geobacter sulfurreducens Contains Separate C- and A-Adding tRNA Nucleotidyltransferases and a Poly(A) Polymerase

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

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