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1 Department of Biology, Emory University, Atlanta, GA 30322, USA
2 Department of Molecular Microbiology, The John Innes Centre, Norwich NR4 7UH, UK
Correspondence
George H. Jones
george.h.jones{at}emory.edu
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ABSTRACT |
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Present address: Pharmazeutische Biologie, Pharmazeutisches Institut, Universität Tübingen, 72076, Tübingen, Germany.
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INTRODUCTION |
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; Rauhut & Klug, 1999; Sarkar, 1996, 1997). In Escherichia coli, polyadenylation targets RNAs for degradation (Carpousis et al., 1999; Régnier & Arraiano, 2000). E. coli cells contain at least two enzymes capable of adding poly(A) tails to the 3'-ends of RNA molecules. The major polyadenylating enzyme, designated poly(A) polymerase I (PAP I, EC 2.7.7.19), was originally identified by virtue of its role in regulating plasmid copy number, and is a product of the pcnB gene (Cao & Sarkar, 1992; He et al., 1993). Mutants of E. coli lacking PAP I still retain the ability to polyadenylate RNAs, indicating that there is at least one other polyadenylating enzyme in those cells (Kalapos et al., 1994). That enzyme does not appear to be the product of the f310 gene designated initially as PAP II, since overexpression of f310 led neither to an increase in PAP activity in the relevant cells nor to an increase in the level of RNA polyadenylation (Mohanty & Kushner, 1999a). Rather, the second PAP in E. coli is polynucleotide phosphorylase (PNPase, EC 2.7.7.8), which normally functions as a 3'-5'-exoribonuclease that catalyses the phosphorolysis of RNA chains, but under appropriate conditions in vivo, can degrade RNAs or synthesize poly(A) tails. PNPase is also 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 (Mohanty & Kushner, 2000b). The overall model for RNA degradation that has emerged from the studies in E. coli involves the generation of RNA 3'-ends via endonucleolytic cleavage by the endoribonucleases RNase E and RNase III, polyadenylation of the 3'-ends by PAP I (and under some conditions by PNPase) and degradation of the resulting RNAs exonucleolytically by PNPase (with the assistance of the DEAD box helicase RhlB), RNase II and RNase R (Cheng & Deutscher, 2005; Coburn & Mackie, 1999; Mohanty & Kushner, 1999b, 2000a; O'Hara et al., 1995). This model applies especially to structured RNAs, with hairpins at their 3'-ends (Carpousis et al., 1999; Régnier & Arraiano, 2000).
As a first step toward understanding the mechanisms of RNA degradation in streptomycetes, we demonstrated that a fraction of total RNA from Streptomyces coelicolor could be bound by oligo(dT) cellulose and that this RNA fraction had the properties expected of RNA that was polyadenylated at its 3'-end (Bralley & Jones, 2001). Moreover, an enzyme activity capable of adding A residues to acceptor RNAs was demonstrated in S. coelicolor mycelial extracts. Unlike the situation in most eukaryotes, we observed that 16S and 23S (but not 5S) rRNAs as well as mRNAs were modified at their 3'-ends in S. coelicolor (Bralley & Jones, 2001). Most interestingly, when the nature of the 3'-tails associated with S. coelicolor RNAs was examined by cDNA cloning and DNA sequencing, it was observed that those tails were heteropolymeric, containing G, C and U as well as A residues (Bralley & Jones, 2002). Heteropolymeric tails were also observed at the 3'-ends of RNAs from Streptomyces antibioticus and we therefore believe that such tails are likely to be found in all streptomycetes (Bralley & Jones, 2003).
The presence of heteropolymeric tails at the 3'-ends of streptomycete RNAs raises the question of the nature of the enzyme responsible for synthesizing those tails. There are at least three candidates for that activity, based on the published sequence of the S. coelicolor genome (Bentley et al., 2002). First, SCO3896 encodes a protein with significant sequence similarity to E. coli PAP I (Bralley & Jones, 2001). A second candidate PAP enzyme is RNase PH (EC 2.7.7.56: candidate gene SCO2904). Like PNPase, RNase PH is a 3'-5'-exoribonuclease that functions phosphorolytically (Deutscher et al., 1988). RNase PH is involved in tRNA maturation in E. coli (Deutscher et al., 1988), Bacillus subtilis (Wen et al., 2005) and presumably in other bacteria as well. Because it is capable of polymerizing nucleoside diphosphates (Ost & Deutscher, 1990), it could conceivably function as a PAP, in the same way that PNPase functions in E. coli. Finally, PNPase (candidate gene SCO5737) might catalyse the formation of RNA 3'-tails in Streptomyces as it does in pcnB mutants of E. coli.
In this report we confirm the results of Sohlberg et al. (2003) that the product of SCO3896 is not a PAP but is rather a tRNA nucleotidyltransferase (TNT), an enzyme that adds CCA to the ends of immature or damaged tRNAs. Moreover, we provide evidence that SCO3896 is an essential gene. We show further that SCO2904 encodes an RNase PH that like, polynucleotide phosphorylase, can polyadenylate RNA 3'-ends in vitro. However RNase PH appears to play no role in the synthesis or maintenance of poly(A) tails in S. coelicolor in vivo. We conclude, therefore, that the most likely candidate for the RNA 3'-polyribonucleotide polymerase in S. coelicolor is polynucleotide phosphorylase. PNPase is essential in S. coelicolor, presumably because of the critical role it plays in RNA degradation. The tRNA nucleotidyltransferase is also essential in S. coelicolor.
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METHODS |
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with the exception that 1 % carboxymethyl cellulose (Sigma) replaced PEG 6000. E. coli strains were grown in LB medium supplemented with antibiotics as necessary.
Cloning of SCO3896 and SCO2904.
SCO3896 and SCO2904 were cloned as PCR products using the primers listed in Table 1. In each case the forward primer used to amplify the gene replaced the GTG start codon annotated in the S. coelicolor genome database with ATG and the reverse primer introduced a BamHI site downstream of the translation stop codon. The templates used were S. coelicolor cosmids H24 (SCO3896 is orf 18 on the cosmid) and E19A (SCO2904 is orf 04c on the cosmid). Conditions for PCR employed a 5 min hot start at 95 °C followed by 35 cycles consisting of denaturation at 95 °C for 1 min, annealing for 2 min at 55 °C and extension for 1 min at 72 °C. The last cycle was followed by a 10 min extension at 72 °C. The PCR products were purified by gel electrophoresis and the bands corresponding to SCO3896 and SCO2904 were isolated from the gel and digested with NdeI and BamHI. The SCO3896 fragment was ligated to the overexpression plasmid pET19B (Novagen) and the SCO2904 fragment was ligated to pET11A; in each case the plasmid had been digested with NdeI and BamHI. Ligation mixtures were used to transform E. coli DH5
and transformants were screened by analysis of plasmid minipreps. The plasmids obtained by this analysis were designated pJSE3550 (SCO3896) and pJSE450 (SCO2904). A sample of each plasmid was used to transform the E. coli overexpression strain BL21(DE3)pLysS (Novagen).
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) and the resulting extract was applied to a 0·9x15 cm column of Source Q (Amersham Biosciences). The column was eluted at room temperature with a gradient of 0–0·3 M NaCl in buffer I and fractions containing the overexpressed protein were identified by SDS-PAGE of 20 µl portions of every third fraction. Relevant fractions were pooled, brought to 1·5 M NaCl by the addition of 5 M NaCl and applied to a 1·2x12 cm column of Phenyl Sepharose CL-4B (Amersham Biosciences) equilibrated with buffer I containing 1·5 M NaCl. The column was washed successively at 4 °C with 50 ml buffer I/1·5 M NaCl, 50 ml buffer I/0·5 M NaCl and 100 ml buffer I. The enzyme began to elute in the 0·5 M NaCl wash and fractions containing SCO2904 were pooled and concentrated by dialysis against ammonium sulfate without stirring (Schreier et al., 1977). PNPase from S. coelicolor was prepared as described previously (Jones et al., 2003).
Assays for PNPase, RNase PH, TNT and PAP.
PNPase polymerization and phosphorolysis assays were performed essentially as described previously (Jones et al., 2003) except that reaction mixtures contained 50 mM KCl. Phosphorolysis assays contained 1·97 mM total (3H-labelled plus unlabelled) poly(A). RNase PH assays were performed using the same conditions as those for PNPase. In both assays, mixtures were incubated for 15 min at 37 °C and [3H]ADP incorporation or the loss of acid-insoluble radioactivity was monitored by TCA precipitation and liquid scintillation counting. In some cases assay mixtures contained 150 µg E. coli tRNA. Results are expressed as µmol ADP incorporated or released based on the total amount of ADP (radioactive and non-radioactive) present in reaction mixtures.
TNT assays (60 µl) contained 50 mM Tris/HCl, pH 8·5; 10 mM MgCl2; 0·2 mM nonradioactive ATP or CTP; 20 µCi (740 kBq) ml–1 of -[32P]ATP or CTP (3000 Ci mmol–1, 110 TBq mmol–1; Amersham Biosciences); 100 µg bovine serum albumin ml–1; 2 mg E. coli tRNA (Sigma) ml–1. Reaction mixtures were incubated for 15 min at 37 °C, after which 50 µl samples were removed and the reaction products were collected by precipitation with tricholoroacetic acid. Precipitates on glass fibre filters were analysed by liquid scintillation counting. E. coli PAP I was obtained from Ambion and assays were performed according to the supplier's instructions using tRNA as acceptor. In some experiments, reaction products from the TNT and PAP reactions were isolated by phenol extraction and ethanol precipitation and analysed on 10 % denaturing polyacrylamide gels.
In other experiments, PNPase, RNase PH and PAP I were incubated with an RNA fragment derived from the readthrough transcript of the rpsO–pnp operon of S. coelicolor. 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 -[32P]CTP, and the 3'-end of the RNase III digestion product represents the primary RNase III processing site in the rpsO–pnp intergenic region. Details of the processing of the rpsO–pnp transcript and the description of the fragments produced have been published elsewhere (Chang et al., 2005). For the studies presented here, polymerization reaction mixtures were prepared as described above for PNPase, RNase PH and PAP I, except that tRNA was omitted. Instead, those mixtures contained 15 000 c.p.m. of the 333 base RNA fragment. Labelled RNAs were isolated from reaction mixtures by phenol extraction and ethanol precipitation and were examined by electrophoresis on 5 % denaturing polyacrylamide gels along with Ambion Century-Plus size markers that were end-labelled using [32P]pCp and T4 RNA ligase.
Analysis of oligo(A) stretches and RNA 3'-tail length and composition.
Total RNA was prepared from S. coelicolor mycelium as described previously (Hsieh & Jones, 1995) and labelled at the 3'-end with [32P]pCp and T4 RNA ligase (Bralley & Jones, 2001, 2003). Labelled RNAs were digested with RNases A and T1 and the digestion products were displayed on sequencing gels. This combination of RNases cleaves all phosphodiester bonds except those between adjacent A residues. End-labelled oligo(dT)18 was used as a size standard in these experiments.
To determine RNA 3'-tail composition RT-PCR was carried out using 10 µg total RNA as described previously (Bralley & Jones, 2002). Briefly, the oligo(dT)17 primer, ADoT, listed in Table 1
, was used for reverse transcription. A single round of PCR was utilized to amplify cDNA of 16S rRNA using the gene-specific forward primer 16S1 and the reverse primer AD20 (Table 1). Two rounds of semi-nested PCR were conducted using the gene-specific primers PNP2 and PNP3 (Table 1) in conjunction with primer ADoT. RT-PCR products were cloned using the TOPO-TA Cloning System (Invitrogen) and candidate clones were sequenced by the Emory University Sequencing Facility.
Gene disruptions.
SCO2904, SCO3896 and SCO5737 were deleted by gene replacement using the PCR targeting method developed by Gust et al. (2003). A disruption cassette containing a resistance marker and an oriT (RK2) for efficient intergeneric conjugation was amplified by PCR with primers E19A.04.forw and E19A.04.rev (disruption of SCO2904), H24.18.forw and H24.18.rev (disruption of SCO3896), and 3C3.23.forw and 3C3.23.rev (disruption of SCO5737, Table 1) respectively. The PCR products were integrated into the corresponding cosmids by 
Red-mediated recombination. The cosmids were conjugated from E. coli ET12567/pUZ8002 to S. coelicolor, where they readily undergo homologous recombination.
To create a strain containing a second copy of SCO3896, a 2281 bp EcoRI–StuI fragment of cosmid H24, bearing SCO3896, was cloned in pSET
. In pSET the apramycin-resistance gene of pSET152 (Bierman et al., 1992) is replaced with the spectinomycin/streptomycin-resistance marker (O'Connor et al., 2002). The resulting construct was transferred to S. coelicolor M145 by conjugation from E. coli and its integration was verified by PCR and Southern blotting. The resulting strain was then subjected to gene disruption using the REDIRECT protocol (Gust et al., 2003). To add a second copy of pnp to S. coelicolor M145, an approximately 3·1 kb PCR fragment, bearing rpsO–pnp, was prepared using cosmid 3C3 as a template. The PCR product was cloned into pSET152 as an XbaI fragment; that fragment was inserted into pSET and the resulting construct was transferred to S. coelicolor M145 by conjugation.
Miscellaneous methods.
Protein was determined by the method of Bradford (1976) using the Bio-Rad assay reagent with bovine serum albumin as a standard. SDS-PAGE was performed essentially as described by Laemmli (1970) and gels were stained with Coomassie brilliant blue.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, lane 3, IPTG induction led to the synthesis of a protein of the expected size (26 400 Da) and essentially homogeneous protein was obtained by the purification procedure described in Methods (Fig. 1, lane 4). Fig. 2 shows the results of polymerization and phosphorolysis assays of the SCO2904 gene product as compared with S. coelicolor PNPase. It is apparent that SCO2904 catalyses both the polymerization of ADP into an acid-insoluble product and the phosphorolysis of poly(A). Added tRNA had no effect on the polymerization activities of either PNPase or SCO2904. Based on its size, sequence similarity to the E. coli enzyme and enzymic activities, we conclude that SCO2904 encodes an RNase PH. The data of Fig. 2 indicate that the S. coelicolor RNase PH is less active than PNPase on a molar basis in both the polymerization and phosphorolysis reactions. This observation may reflect the fact that RNase PH is thought to play a specific role in tRNA maturation in bacteria as opposed to the more general function of PNPase as a 3'-5'-exoribonuclease (Deutscher, 1995). Thus, RNase PH might not be expected to function as effectively as PNPase in phosphorolysis or polymerization with poly(A) and ADP, respectively, as substrates.
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). PAPs and TNTs, the enzymes that add the CCA to the 3'-termini of immature and damaged tRNAs, belong to the same superfamily of nucleotidyltransferases (Holm & Sander, 1995; Yue et al., 1996), so the SCO3896 gene product is also similar in sequence to the E. coli TNT (38 % identity, 48 % similarity), and it is even more similar to PAPS from B. subtilis (43 % identity, 53 % similarity). It was shown some years ago that PAPS is not the B. subtilis poly(A) polymerase; rather it is a tRNA nucleotidyltransferase (Raynal et al., 1998). Sohlberg et al. (2003) have also shown that SCO3896 is a TNT and we have reached the same conclusion by overexpressing and purifying the gene product (Fig. 1, lanes 1 and 2) and verifying its activity. Fig. 3A shows that SCO3896 was capable of adding C and A residues to tRNA acceptors (lanes 2 and 3) but did not synthesize the high-molecular-mass species that would be produced by a poly(A) polymerase (lane 4). It should be noted that the substrates for A and C addition in the total RNA pool used as the substrate in these assays are presumably species lacking complete CCA ends.
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. As expected, PAP I, in the presence of ATP, converted the 333 base fragment to larger species, some of which were over 1000 bases in length (Fig. 3B, lane 5). Similarly, PNPase, when incubated with the 333 base fragment and ADP, converted the fragment into higher-molecular-mass products (Fig. 3B, lane 3). Fig. 3B, lane 4, shows that RNase PH also has this polymerizing activity. The 333 base substrate was converted to species at least 750 bases in total length. Thus, RNase PH, like PNPase, can function as a poly(A) polymerase in vitro. To our knowledge this is the first demonstration that RNase PH has poly(A) polymerase activity with a naturally occurring substrate other than tRNA.
RNase PH is not the S. coelicolor PAP
Given the observation that S. coelicolor RNase PH can function as a PAP in vitro it was of considerable interest to examine the potential role of this enzyme in the synthesis of RNA 3'-tails in vivo. To this end, SCO2904 was disrupted by the insertion of a viomycin-resistance cassette using the REDIRECT gene disruption protocol (Gust et al., 2003). Disruption of the gene was confirmed by PCR and by Southern blotting (data not shown), demonstrating insertion of the viomycin-resistance cassette into SCO2904. RNA was prepared from S. coelicolor M145 and from two putative disruptant strains, JSE450A and 450B, and the 3'-tails associated with the RNA species in those preparations were analysed for oligo(A) stretches embedded in the heteropolymeric 3-tails (Fig. 4). It can be seen that the distribution of those stretches is identical in the parent strain, S. coelicolor M145, and in the two disruptant strains. Prominent bands representing oligo(A) stretches of 1–18 residues are observed in the preparations from the parent strain and both disruptant strains. Overexposure of the autoradiogram shown in Fig. 4 revealed bands in all three preparations that were 25–35 residues in length. This observation confirms previous results that while oligo(A) stretches of less than 18–20 residues are most commonly observed as components of the heteropolymeric 3'-tails of streptomycete RNAs, longer stretches do occur at a lower frequency (Bralley & Jones, 2003). It should be noted that because the poly(A) tails of streptomycete RNAs are highly heteropolymeric (Bralley & Jones, 2002), the pattern of continuous increase in tail length observed in E. coli (lane 3) is not observed for the tails from S. coelicolor.
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, revealed no significant difference in tail length or composition of cDNAs corresponding to the polyadenylated RNAs isolated from wild-type or mutant strains. The tails of 16S rRNA appeared similar to 16S rRNA tails isolated from S. coelicolor M145 (Bralley & Jones, 2002). These experiments generated a variety of cDNAs from genes possessing unanticipated homology to the primer (PNP3) used in RT-PCR. These cDNAs, from leuA (SCO0387), clpP (SCO3044) and a 23S rRNA, were isolated from both wild-type and mutant strains. These clones revealed tails with compositions similar to those previously isolated from genes actII-orf4 and redD (Bralley & Jones, 2002) and S. antibioticus pnp (Bralley & Jones, 2003). The fact that both RNA tail length and composition in the mutant JSE450A appear similar to that found in M145 strongly suggests that RNase PH is not involved in the synthesis of RNA 3'-tails in S. coelicolor.
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, 2002, 2003). Attempts were also made, as a part of the studies reported here, to disrupt SCO3896 and pnp (SCO5737) using a PCR-targeted mutagenesis approach that yields high frequencies of gene disruption in Streptomyces (Gust et al., 2003). To this end, an apramycin-resistance cassette was synthesized by PCR with 5'- and 3'-ends that correspond to regions flanking either SCO3896 or SCO5737. These cassettes were inserted into the corresponding cosmids and the resulting E. coli strains bearing the mutagenized cosmids were mixed with spores of S. coelicolor M145 to allow conjugal transfer of the cosmids. Subsequent homologous recombination would be expected to result in replacement of the chromosomal copy of SCO3896 or SCO5737 with the disrupted copy at high frequencies. More than 1000 exconjugant colonies were screened from each conjugation by PCR, but they all contained both a disrupted and an intact copy of SCO3896 and SCO5737, respectively. No double crossover replacements were observed.
This result suggested that both SCO3896 and SCO5737 are essential in S. coelicolor. To test this possibility, a copy of each gene was cloned into the integrative plasmid vector pSET and introduced into M145. pSET contains the bacteriophage 
C31 attP site and integrates into the attB site in the S. coelicolor chromosome (Bierman et al., 1992). The insertions into the S. coelicolor genome were verified by Southern blotting and the attempts to disrupt SCO3896 and SCO5737 were repeated. In the case of SCO3896, more than 30 % of the exconjugants obtained were double crossovers and approximately 50 % double crossovers were observed in the case of SCO5737. Both PCR using primers specific for each gene, and Southern blotting, confirmed that the original chromosomal copy had been successfully disrupted in some of those exconjugants. Thus, it was possible to disrupt SCO3896 or SCO5737 only after inserting a second copy of the relevant gene into the chromosome.
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DISCUSSION |
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; Price et al., 1999). Unlike the situation in E. coli, streptomycetes do not appear to contain an RNase II, the 3'-5'- exoribonuclease that functions hydrolytically or RNase R (Zuo & Deutscher, 2001). Thus, the major 3'-5'-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.
Several recent studies of the streptomycete PNPase have confirmed its function as a phosphorolytic exoribonuclease (Jones & Bibb, 1996; Jones et al., 2003). The unanswered question has to do with its function in the synthesis of RNA 3'-tails. The most straightforward way to examine this question would be to disrupt pnp in S. coelicolor and to examine the effects of that disruption on the length and composition of the tails associated with RNAs in the disruptant strains. However, as we demonstrate here, PNPase is an essential gene in S. coelicolor. Thus, we have adopted a ‘process of elimination’ approach to define the role of PNPase in RNA 3'-end synthesis. We have shown that SCO3896, which has significant sequence similarity to E. coli PAP I, is not a poly(A) polymerase but is rather a tRNA nucleotidyltransferase. These findings confirm the results of Sohlberg et al. (2003).
As PNPase would function in its polymerization mode as a polyribonucleotide polymerase, it seemed formally possible that a second enzyme with similar activity, RNase PH, might be responsible for synthesis of RNA 3'-tails in streptomycetes. Indeed we have shown that S. coelicolor RNase PH can polymerize ADP in vitro just as can PNPase. Moreover, we have shown that RNase PH can add 3'-tails to a cellular RNA, again like PNPase. Thus, the analysis of Sohlberg et al. (2003), which was based on their observation that PNPase could function as a PAP in vitro, omitted another enzyme, RNase PH, which has exactly the same in vitro activity. We show here, however, that disruption of the RNase PH gene has no effect on the length or composition of the tails found at the 3'-ends of various cellular RNAs. Thus, we conclude that neither the product of SCO3896 nor that of SCO2904 plays a significant role in the synthesis of the 3'-tails in S. coelicolor. We argue, as do Sohlberg et al. (2003), that the enzyme responsible for the synthesis of those tails is PNPase. This conclusion is consistent with the observation that spinach chloroplasts and a Synechocystis species, like S. coelicolor, do not encode a dedicated PAP (Rott et al., 2003; Yehudai-Resheff et al., 2001). In the two former systems, and in pcnB mutants of E. coli, the RNA 3'-polyribonucleotide polymerase is PNPase and as in S. coelicolor, the 3-tails of RNAs in spinach chloroplasts, in the Synechocystis sp. and in pcnB mutants are heteropolymeric (Mohanty & Kushner, 2000b; Rott et al., 2003; Yehudai-Resheff et al., 2001). We cannot exclude the possibility that RNase PH may add residues infrequently to RNA 3'-tails in vivo, as is the case for PNPase (Mohanty & Kushner, 2000b), but our data clearly indicate that RNase PH is not the PAP in S. coelicolor.
Why should PNPase and TNT be essential in S. coelicolor? In the case of PNPase, the answer would appear to be that it is the only enzyme in streptomycetes capable of catalysing two important reactions involved in RNA degradation: the exonucleolytic removal of 3'-tails (recall that streptomycetes do not contain an RNase II or RNase R) and the synthesis of those same tails. As regards SCO3896, inspection of the S. coelicolor genome reveals that less than a third of the tRNA genes encode the CCA end. Thus, most S. coelicolor tRNAs, many of which must be essential for protein synthesis, require SCO3896 to produce mature 3'-ends. In contrast, the studies of Zhu & Deutscher (1987) show definitively that TNT is not essential in E. coli. Thus, all essential tRNA genes in that organism must encode the CCA end.
We show in another publication that of the known bacterial phyla only the 
- and 
-proteobacteria contain both a TNT and a dedicated PAP (Bralley et al., 2005). We believe it possible, therefore, that PNPase may function as the RNA 3'-polyribonucletide polymerase in other bacterial species. Our recent results suggest, however, that there are at least three biochemical systems for the synthesis of RNA 3'-tails in bacteria. As discussed above, the PAP I homologue in B. subtilis is actually a TNT. Moreover, we have demonstrated poly(A) tail synthesis in B. subtilis mutants lacking both the TNT and PNPase (Campos-Guillén et al., 2005). We are currently examining the enzymology of poly(A) tail formation in B. subtilis.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bierman, M., Logan, R., O'Brien, K., Seno, E. T., Rao, R. N. & Shoner, B. E. (1992). Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene 116, 43–49.[CrossRef][Medline]
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem 72, 248–254.[CrossRef][Medline]
Bralley, P. & Jones, G. H. (2001). Poly(A) polymerase activity and RNA polyadenylation in Streptomyces coelicolor A3(2). Mol Microbiol 40, 1155–1164.[CrossRef][Medline]
Bralley, P. & Jones, G. H. (2002). cDNA cloning confirms the polyadenylation of RNA decay intermediates in Streptomyces coelicolor. Microbiology 148, 1421–1425.
Bralley, P. & Jones, G. H. (2003). Overexpression of the polynucleotide phosphorylase gene (pnp) of Streptomyces antibioticus affects mRNA stability and poly(A) tail length but not ppGpp levels. Microbiology 149, 2173–2182.
Bralley, P. & Jones, G. H. (2004). Organization and expression of the polynucleotide phosphorylase gene (pnp) of Streptomyces: processing of pnp transcripts in Streptomyces antibioticus. J Bacteriol 186, 3160–3172.
Bralley, P., Chang, S. A. & Jones, G. H. (2005). A phylogeny of bacterial nucleotidyltransferases: Bacillus halodurans contains two tRNA nucleotidyltransferases. J Bacteriol 187, 5927–5936.
Campos-Guillén, J., Bralley, P., Jones, G. H., Bechhofer, D. H. & Almeda-Alvarez, G. (2005). Addition of poly(A) and heteropolymeric 3'-ends in Bacillus subtilis wild-type and PNPase-deficient strains. J Bacteriol 187, 4698–4706.
Cao, G. J. & Sarkar, N. (1992). Identification of the gene for an Escherichia coli poly(A) polymerase. Proc Natl Acad Sci U S A 89, 10380–10384.
Carpousis, A. J., Vanzo, N. F. & Raynal, L. C. (1999). mRNA degradation: a tale of poly(A) and multiprotein machines. Trends Genet 15, 24–28.[CrossRef][Medline]
Chang, S. A., Bralley, P. & Jones, G. H. (2005). The absB gene encodes a double-strand specific endoribonuclease that cleaves the readthrough transcript of the rspO-pnp operon in Streptomyces coelicolor. J Biol Chem 280, 33213–33219.
Cheng, Z. F. & Deutscher, M. P. (2005). An important role for RNase R in mRNA decay. Mol Cell 17, 313–318.[CrossRef][Medline]
Coburn, G. A. & Mackie, G. A. (1999). Degradation of mRNA in Escherichia coli: an old problem with some new twists. Prog Nucleic Acids Res Mol Biol 62, 55–105.[Medline]
Deutscher, M. (1995). tRNA processing nucleases. In tRNA: Structure, Biosynthesis and Function, pp. 51–65. Edited by D. Söll & U. L. RajBhandary. Washington, DC: American Society for Microbiology.
Deutscher, M., Marshall, G. T. & Cudny, H. (1988). RNase PH: an Escherichia coli phosphate-dependent nuclease distinct from polynucleotide phosphorylase. Proc Natl Acad Sci U S A 85, 4710–4714.
Gust, B., Challis, G. L., Fowler, K., Kieser, T. & Chater, K. F. (2003). PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc Natl Acad Sci U S A 100, 1541–1546.
Hagège, J. M. & Cohen, S. N. (1997). A developmentally regulated Streptomyces endoribonuclease resembles ribonuclease of Escherichia coli. Mol Microbiol 25, 1077–1090.[CrossRef][Medline]
He, L., Soderbom, F., Wagner, E. G., Binnie, U., Binns, N. & Masters, M. (1993). PcnB is required for the rapid degradation of RNAI, the antisense RNA that controls the copy number of ColE1-related plasmids. Mol Microbiol 9, 1131–1142.[CrossRef][Medline]
Holm, L. & Sander, C. (1995). DNA polymerase beta belongs to an ancient nucleotidyltransferase superfamily. Trends Biochem Sci 20, 345–347.[CrossRef][Medline]
Hsieh, C.-J. & Jones, G. H. (1995). Nucleotide sequence, transcriptional analysis and glucose regulation of the phenoxazinone synthase gene from Streptomyces antibioticus. J Bacteriol 177, 5740–5747.
Jones, G. H. & Bibb, M. J. (1996). Guanosine pentaphosphate synthetase from Streptomyces antibioticus is also a polynucleotide phosphorylase. J Bacteriol 178, 4281–4288.
Jones, G. H., Symmons, M. F., Hankins, J. S. & Mackie, G. A. (2003). Overexpression and purification of untagged polynucleotide phosphorylases. Protein Expr Purif 32, 202–209.[CrossRef][Medline]
Kalapos, M. P., Cao, G. J., Kushner, S. R. & Sarkar, N. (1994). Identification of a second poly(A) polymerase in Escherichia coli. Biochem Biophys Res Commun 198, 459–465.[CrossRef][Medline]
Kieser, Y., Bibb, M. J., Buttner, M. J., Chater, K. F. & Hopwood, D. A. (2000). Practical Streptomyces Genetics. Norwich: John Innes Foundation.
Laemmli, U. (1970). Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef][Medline]
Lee, K. & Cohen, S. N. (2001). A Streptomyces coelicolor functional analog of E. coli RNase E shows evolutionary shuffling of catalytic and PNPase domains. In Proceedings of the 12th International Symposium on the Biology of Actinomycetes, p. 66.
Mohanty, B. K. & Kushner, S. R. (1999a). Residual polyadenylation in poly(A) polymerase I (pcnB) mutants of Escherichia coli does not result from the activity encoded by the f310 gene. Mol Microbiol 34, 1109–1119.[CrossRef][Medline]
Mohanty, B. K. & Kushner, S. R. (1999b). Analysis of the function of Escherichia coli poly(A) polymerase in RNA metabolism. Mol Microbiol 34, 1094–1108.[CrossRef][Medline]
Mohanty, B. K. & Kushner, S. R. (2000a). Polynucleotide phosphorylase, RNase II and RNase E play different roles in the in vivo modulation of polyadenylation in Escherichia coli. Mol Microbiol 36, 982–994.[CrossRef][Medline]
Mohanty, B. K. & Kushner, S. R. (2000b). Polynucleotide phosphorylase functions both as a 3'-5' exonuclease and a poly(A) polymerase in Escherichia coli. Proc Natl Acad Sci U S A 97, 11966–11971.
O'Connor, T. J., Kanellis, P. & Nodwell, J. R. (2002). The ramC gene is required for morphogenesis in Streptomyces coelicolor and expressed in a cell type-specific manner under the direct control of RamR. Mol Microbiol 45, 45–57.[CrossRef][Medline]
O'Hara, E. B., Chekanova, J. A., Ingle, C. A., Kushner, Z. R., Peters, E. & Kushner, S. R. (1995). Polyadenylation helps regulate mRNA decay in Escherichia coli. Proc Natl Acad Sci U S A 92, 1807–1811.
Ost, K. A. & Deutscher, M. (1990). RNase PH catalyzes a synthetic reaction, the addition of nucleotides to the 3'-ends of RNA. Biochimie 72, 813–818.[Medline]
Price, B., Adamis, T. & Champness, W. (1999). A Streptomyces coelicolor antibiotic regulatory gene, absB, encodes an RNase III homolog. J Bacteriol 181, 6142–6151.
Rauhut, R. & Klug, G. (1999). mRNA degradation in bacteria. FEMS Microbiol Rev 23, 353–370.[CrossRef][Medline]
Raynal, L. C., Krisch, H. M. & Carpousis, A. J. (1998). The Bacillus subtilis nucleotidyl transferase is a tRNA CCA-adding enzyme. J Bacteriol 180, 6276–6282.
Régnier, P. & Arraiano, C. M. (2000). Degradation of mRNA in bacteria: emergence of ubiquitous features. Bioessays 22, 235–244.[CrossRef][Medline]
Rott, R., Zipor, G., Portnoy, V., Liveanu, V. & Schuster, G. (2003). RNA polyadenylation and degradation in cyanobacteria are similar to the chloroplast but different from E. coli. J Biol Chem 278, 15771–15777.
Sarkar, N. (1996). Polyadenylation of mRNA in bacteria. Microbiology 142, 3125–3133.[Medline]
Sarkar, N. (1997). Polyadenylation of mRNA in prokaryotes. Annu Rev Biochem 66, 173–197.[CrossRef][Medline]
Schreier, M. H., Erni, B. & Staehlin, T. (1977). Initiation of mammalian protein synthesis. I. Purification and characterization of seven initiation factors. J Mol Biol 116, 727–753.[CrossRef][Medline]
Sohlberg, B., Huang, J. & Cohen, S. N. (2003). The Streptomyces coelicolor polynucleotide phosphorylase homologue, and not the putative poly(A) polymerase can polyadenylate RNA. J Bacteriol 185, 7273–7278.
Wen, T., Oussenko, I. A., Pellegrini, O., Bechhofer, D. H. & Condon, C. (2005). Ribonuclease PH plays a major role in the exonucleolytic maturation of CCA-containing tRNA precursors in Bacillus subtilis. Nucleic Acids Res 33, 3636–3643.
Yehudai-Resheff, S., Hirsh, M. & Schuster, G. (2001). Polynucleotide phosphorylase functions as both an exonuclease and a poly(A) polymerase in spinach chloroplasts. Mol Cell Biol 21, 5408–5416.
Yue, D., Maizels, N. & Weiner, A. M. (1996). CCA-adding enzymes and poly(A) polymerases are all members of the same nucleotidyltransferase superfamily: characterization of the hyperthermophile Sulfolobus shibatae. RNA 2, 895–908.[Abstract]
Zhu, L. & Deutscher, M. P. (1987). tRNA nucleotidyltransferase is not essential for Escherichia coli viability. EMBO J 6, 2473–2477.[Medline]
Zuo, Y. & Deutscher, M. (2001). Exoribonuclease superfamilies: structural analysis and phylogenetic distribution. Nucleic Acids Res 29, 1017–1026.
Received 21 July 2005;
revised 14 October 2005;
accepted 1 November 2005.
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) and RNase PH (
). Polymerization assays were performed using [3H]ADP as the substrate and phosphorolysis assays using [3H]poly(A) as the substrate. Values are the mean of duplicate assays that differed by no more than 10 %, after subtracting a zero-time control from each experimental data point. In some experiments reaction mixtures contained 150 µg E. coli tRNA ml–1.
