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Originally published In Press as doi:10.1074/jbc.M503440200 on August 1, 2005

J. Biol. Chem., Vol. 280, Issue 39, 33213-33219, September 30, 2005
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The absB Gene Encodes a Double Strand-specific Endoribonuclease That Cleaves the Read-through Transcript of the rpsO-pnp Operon in Streptomyces coelicolor*

Samantha A. Chang, Patricia Bralley, and George H. Jones1

From the Department of Biology, Emory University, Atlanta, Georgia 30322

Received for publication, March 29, 2005 , and in revised form, July 20, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The absB locus of Streptomyces coelicolor encodes a homolog of bacterial RNase III. We cloned and overexpressed the absB gene product and purified a decahistidine-tagged version of the protein. We show here that AbsB is active against double-stranded RNA transcripts derived from synthetic DNAs but is inactive with single-stranded homopolymers. We thus designate the absB product RNase IIIS. Using T7 RNA polymerase and a cloned template containing the rpsO-pnp intergenic region, we synthesized an RNA substrate representing a portion of the read-through transcript normally produced in S. coelicolor. This transcript contains the sequences that form the putative rpsO terminator and those that form an intergenic stem-loop structure thought to be the site for RNase IIIS processing of the read-through transcript. We show that RNase IIIS does cleave that model transcript, with primary and secondary cleavage sites in an internal loop in the stem-loop structure. We have identified the primary and secondary cleavage sites by primer extension and demonstrate the further processing of the initial cleavage products. Thus, as is the case in Escherichia coli, the read-through transcript from rpsO-pnp is cleaved by RNase IIIS in S. coelicolor. However, the cleavage sites are different in the two systems. The positions of the cleavage sites in the stem-loop of the S. coelicolor transcript are more akin to those identified in the processing of bacteriophage T7 mRNAs. A kinetic assay for RNase IIIS was developed, and kinetic parameters for the reaction utilizing the model transcript from rpsO-pnp were determined.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ribonuclease III is a double strand-specific endoribonuclease found in bacteria and eukaryotes. In Escherichia coli, RNase III is involved in the processing and maturation of viral, ribosomal, and messenger RNAs. Studies of the processing of bacteriophage T7 RNAs demonstrated an important role for RNase III and identified sequence features that determine the specificity of cleavage (1–3). RNase III is involved in the processing of the 30 S ribosomal RNA precursor to pre-16 S and pre-23 S intermediates in E. coli, and the 30 S precursor only accumulates in RNase III-deficient mutants (4, 5). Of particular relevance to the present study, RNase III is involved in the processing of transcripts from the rpsO-pnp operon in E. coli. The operon is transcribed from two promoters, PrpsO, situated upstream of rpsO, the gene for ribosomal protein S15, and Ppnp, a promoter located in the intergenic region between rpsO and pnp, the gene for polynucleotide phosphorylase (6). Transcription initiating at PrpsO produces a read-through transcript containing both rpsO and pnp, whereas initiation at Ppnp produces a pnp transcript only (6). Secondary structure models and biochemical studies have confirmed the presence of a stem-loop structure in the rpsO-pnp intergenic region, upstream of the pnp translation start site (6, 7). This stem loop is present in both the read-through transcript and the transcript from Ppnp and is cleaved at identical sites in the two transcripts by RNase III (6, 7). RNase III cleavage of the pnp transcripts induces PNPase2 autoregulation of pnp expression by an as yet unknown mechanism that may involve PNPase binding to the processed mRNAs to prevent their translation (7–9).

We have recently begun a study of pnp expression in Streptomyces. As in E. coli, pnp is transcribed from two promoters to produce an rpsO-pnp read-through transcript and a transcript initiating at an intergenic promoter, Ppnp (10). The organization of the operon is conserved in Streptomyces antibioticus, Streptomyces coelicolor, and Streptomyces avermitilis (10). Computer modeling predicted the formation of two stem loops or hairpins in the S. antibioticus and S. coelicolor rpsO-pnp read-through transcripts. We argued that the leftmost hairpin is the rpsO terminator, whereas the rightmost is the locus for ribonuclease processing of the read-through transcript (10). In E. coli, Ppnp is situated between the rpsO terminator and the processed hairpin, allowing that hairpin to form in both the read-through transcript and the transcript from Ppnp (6). In S. antibioticus, the –10 region of Ppnp is situated in the stem of the rightmost hairpin, thought to be the processing site. Thus, transcription from Ppnp does not produce a structure that is an obvious substrate for RNase III. Rather, computer modeling predicted a cloverleaf structure with several single-stranded regions (10). Using primer extension and RNA ligase mediated reverse transcription-PCR, we identified a number of putative processing sites in the rpsO-pnp intergenic region of S. antibioticus. Our data suggest that, unlike the situation in E. coli, only the read-through transcript in Streptomyces is processed by RNase III cleavage, whereas the transcript from Ppnp may be processed by a single strand-specific endonuclease like RNase E (10).

To examine further the processing of the Streptomyces rpsO-pnp transcripts, we have overexpressed and purified the product of the absB locus of S. coelicolor (11) and show here that it is a double strand-specific endoribonuclease. We have synthesized a template corresponding to the rpsO-pnp intergenic region from S. coelicolor and have identified RNase III cleavage sites in that transcript. Those cleavage sites occur in a different position in the stem-loop as compared with the sites in the rpsO-pnp transcripts in E. coli, and these results coupled with our earlier observations (10) suggest that the processing of rpsO-pnp transcripts in Streptomyces differs in important respects from the corresponding process in E. coli. Our in vitro data are consistent with those obtained in vivo (10) and summarized above for the processing of the rpsO-pnp intergenic region in S. antibioticus.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid Construction and Bacterial Strains—To express the AbsB protein, an 819-bp fragment corresponding to the absB open reading frame from S. coelicolor (12) was amplified by the PCR using primers SCABSBF2 and SCABSBR1 (TABLE ONE) with S. coelicolor chromosomal DNA as the template. The forward primer changed the absB start codon from GTG to ATG and introduced an NdeI site at the 5'-end of the PCR product and the reverse primer introduced a BamHI site just downstream of the absB stop codon. The fragment was cloned into pET11A (Novagen) and the resulting construct was designated pJSE1801. This plasmid was digested with NdeI and BamHI and the insert ligated to pET19b (Novagen) to create pJSE1811.


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  		TABLE ONE
Primers used in this study

 
To construct the plasmid used to synthesize the rpsO-pnp transcript, a 459-bp fragment of the rpsO-pnp intergenic region of S. coelicolor was amplified by the PCR from the plasmid pJSE600 using primers rps1 and +16R (TABLE ONE). pJSE600 contains a ~3.1-kb fragment representing the entire rpsO-pnp operon of S. coelicolor. The PCR fragment was cloned into pCR2.1-TOPO (Invitrogen) following the manufacturer's protocol and the construct was designated pJSE5600. Plasmids were verified for correct orientation relative to the T7 promoter by restriction digestion and sequencing.

Expression and Purification of His-tagged AbsB—A 500-ml culture of BL21 (DE3) pLysS/pJSE1811 was grown at 37 °C with shaking in Luria-Bertani medium, supplemented with 50 µg/ml carbenicillin and 34 µg/ml of chloramphenicol to an A600 of 0.5–0.7, and induced with 1 mM isopropyl-{beta}-D-thiogalactoside for 3 h. Cells were harvested by centrifugation, and the pellet was stored at –20 °C for at least 45 min. Cells were thawed by resuspending in 20 ml of ice-cold lysis buffer (50 mM sodium phosphate (pH 8.0), 300 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, and 0.75 mg/ml lysozyme), incubated on ice for 60 min, sonicated briefly, and then centrifuged twice at 10,000 x g for 20 min at 4 °C. Purification was performed using the batch/gravity flow column method as described in the Talon immobilized metal affinity chromatography protocol (BD Biosciences Clontech), except that all washes were conducted at 4 °C and buffers were used at pH 8.0. Fractions from the Talon column were analyzed by SDS-PAGE, tested for activity using an RNase III cleavage assay (see below), pooled, and sequentially dialyzed against Buffer 1 (50 mM Tris-HCl (pH 8.0), 1 M NaCl, 150 mM imidazole, 5 mM MgCl2), Buffer 2 (same as Buffer 1, except without imidazole), and finally overnight against Buffer 3 (50 mM Tris-HCl (pH 8.0), 1 M NaCl, 1 mM EDTA, 1 mM dithiothreitol, 5 mM MgCl2, 50% glycerol). Buffers were supplemented with 2 mM phenylmethylsulfonyl fluoride and one Roche Complete protease inhibitor mixture tablet (Roche Applied Science) per 250 ml. Enzyme was stored in Buffer 3 at –20 °C.

Synthesis of Double-stranded Poly(G)-Poly(C) and Poly(A)-Poly(U) Substrates—Synthetic double-stranded substrates were prepared in 600-µl reactions containing 400 mM Tris-HCl (pH 7.5); 100 mM MgCl2; 1mM EDTA; 1.5 M KCl; 150 µM each ATP, UTP, CTP, and GTP; 0.1 mM dithiothreitol; 50 µg/ml bovine serum albumin; 90 µg of either poly(dG)-poly(dC) or poly(dA)-poly(dT) (Amersham Biosciences); 100 µCi of either [{alpha}-32P]CTP or [{alpha}-32P]ATP (3,000 Ci/mmol; Amersham Biosciences); and 37.5 units of E. coli RNA polymerase (Sigma). Reactions were incubated for 3 h at 37°C. After incubation, an equal volume of TSE buffer (50 mM Tris-HCl (pH 7.0), 100 mM NaCl, 1 mM EDTA) was added. This mixture was extracted with an equal volume of phenol, and then an equal volume of ethanol was added to the aqueous layer. The solution was purified over a 2.0-ml column of Whatman CF11 cellulose as described (13, 14). Fractions containing double-stranded RNA were identified by liquid scintillation counting and pooled. Single-stranded substrates were prepared using only a single NTP in the transcription reactions and were collected by phenol extraction of reaction mixtures and ethanol precipitation.

Synthesis of in Vitro Transcripts of the rpsO-pnp Intergenic Region—His-tagged T7 RNA polymerase was expressed from pBH161 as described (15) but purified using the Talon IMAC resin (BD Biosciences Clontech) as described above. pJSE5600 was linearized with BamHI and purified using the Qiagen PCR purification kit. Transcription reactions (20 µl) contained 30 mM Hepes (pH 7.8); 100 mM potassium glutamate; 15 mM magnesium acetate; 0.25 mM EDTA; 1 mM dithiothreitol; 0.05% Tween 20; 2 mM each ATP, CTP, GTP, and UTP; 1 µg of linearized plasmid DNA; 500 ng of His-tagged T7 RNA polymerase; and 40 units of RNasin (Promega) (15, 16). Reactions were preheated for 2 min at 37 °C before adding RNA polymerase and then incubated at 37 °C for 4 h. Large scale transcription reactions were scaled proportionately up to 1 ml in order to produce 1-mg amounts of transcribed RNA. After transcription, the reaction was supplemented with CaCl2 to a concentration of 1.5 mM and digested twice with 1 unit of DNase I (Promega) per µgof DNA for 30 min at 37 °C. Mixtures were then extracted twice with phenol/chloroform/isoamyl alcohol, followed by extraction with chloroform and precipitation with one-third volume of 10 M ammonium acetate and 2 volumes of ethanol. The RNA was resuspended in water and stored at –20 °C.

For internally labeled transcripts, 1 µCi of [{alpha}-32P]CTP was added per 20 µl of transcription reaction. In some experiments, transcripts were 3'-end-labeled using [32P]pCp and T4 RNA ligase (Ambion). Unlabeled RNA that had been dephosphorylated with calf intestine alkaline phosphatase (Promega) was 5'-end-labeled using [{gamma}-32P]ATP and T4 polynucleotide kinase (Promega). All radioisotopes had a specific activity of 3,000 Ci/mmol and were obtained from Amersham Biosciences. The full-length in vitro transcription product from linearized pJSE5600 is 573 bases in length and includes 72 bases of pCR2.1-TOPO vector sequence at its 5'-end and 42 bases of vector sequence at the 3'-end, downstream of the 459-base rpsO-pnp insert (See Fig. 3).

RNase III Cleavage Assays—Cleavage assays were performed in 60-µl reactions containing 30 mM Tris-HCl (pH 8.0), 100 mM KCl, 0.1 mM EDTA, 0.1 mM dithiothreitol, 10 mM MgCl2, and 50 µg/ml bovine serum albumin. 32P-Labeled double- or single-stranded RNAs (3,000–6,000 cpm) were digested with increasing concentrations of purified AbsB (see Fig. 2; 100 ng of AbsB in a 60-µl reaction mixture is 52.7 nM). After a 10-min incubation at 37 °C, 50 µl of each sample was spotted onto Whatman GF/C glass fiber filter papers, which were washed twice with 10% trichloroacetic acid, 0.6% casamino acids, 0.4% sodium pyrophosphate and once with 70% ethanol. Papers were dried and counted by liquid scintillation. Activity was expressed as a percentage of the original amount of acid-insoluble radioactivity released in the incubation.

Cleavage assays of the rpsO-pnp in vitro transcripts were performed in 10-µl reactions containing the same cleavage buffer described above and 0–200 ng of enzyme. Mixtures typically contained 3 µg of transcript (~1.6 µM, 10,000–40,000 cpm). Reactions were incubated for 20 min at 37 °C and terminated by the addition of 10 µl of stop solution (U.S. Biochemical Corp.). The cleavage products from internally labeled and 3'- and 5'-end-labeled transcripts were separated on a 7 M urea, 5% polyacrylamide gel alongside Ambion RNA Century Plus size markers and visualized by autoradiography.

Kinetic assays were performed as described above except that duplicate reaction mixtures containing 25 ng (79.1 nM) of AbsB and a 0.27–2.7 µM concentration of the 32P-labeled rpsO-pnp transcript were incubated for 2.5 min. Reaction mixtures were applied to 7 M urea, 5% polyacrylamide gels to separate the digestion products. Autoradiograms were scanned, and the densities of bands were quantified using the Scion Image for Windows software. The amounts of RNA in the fragment 1 band (see Fig. 4) produced by AbsB digestion were determined from standard curves generated using the intact 32P-labeled rpsO-pnp transcript and were converted to nmol of product using the calculated molecular weight of fragment 1. Initial velocities were calculated from these data and Lineweaver-Burk plots were used to determine kinetic parameters.

Primer Extension Mapping of Cleavage Site—For each primer extension reaction, 3 µg of internally labeled in vitro transcript were incubated with 0–200 ng of purified AbsB as described above. The reaction mixtures were extracted with phenol/chloroform/isoamyl alcohol and chloroform and precipitated with one-tenth volume of 3 M sodium acetate and 2 volumes of ethanol with the addition of 5 µgof E. coli tRNA as carrier. Primer extension reactions were performed as described previously (10), using primers +16R, Px4, TOPO R1, and TOPO R2. After primer extension, the reactions were treated with 2 units of RNase H (Invitrogen) and separated on a 5.2% sequencing gel. The primers were used with DNA from pJSE5600 to generate the sequencing ladder.

Miscellaneous Methods—Protein concentrations were determined by the Bradford method (17) using the Bio-Rad Protein Reagent and bovine serum albumin as a standard. SDS-PAGE was performed as described by Laemmli (18). All PCRs were performed as follows: 95 °C for 5 min and then 35 cycles at 95 °C for 1 min, 50 °C for 2 min, 72 °C for 1 min, with a final extension at 72 °C for 10 min. The RNA folding program, mfold, version 3.1 was used to predict the secondary structure of the rpsO-pnp in vitro transcripts.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Overexpression, Purification, and Activity Assay of the absB Gene Product—The absB locus of S. coelicolor was identified by Champness and co-workers (11, 19) by mutational analysis and was shown to encode a global regulator of antibiotic production. Complementation of the absB mutation followed by DNA sequencing identified an open reading frame with significant homology to bacterial RNase III (12). Champness and co-workers (12) showed that the 30 S rRNA precursor accumulated in the absB mutants but did not characterize the AbsB activity further. We overexpressed the AbsB protein as a decahistidine-tagged derivative by cloning a PCR fragment in pET19B and purified the enzyme by immobilized metal ion affinity chromatography as described under "Experimental Procedures." As shown in Fig. 1, an essentially homogeneous protein of the expected size, 31,600, was obtained.

We examined the activity of the enzyme using double- and single-stranded RNA substrates prepared using E. coli RNA polymerase with synthetic DNA polymers as templates. Results of AbsB enzyme assays with radiolabeled poly(G)-poly(C) and poly(A)-poly(U) double-stranded templates, purified by cellulose chromatography as described under "Experimental Procedures," are shown in Fig. 2. The enzyme was quite active with poly(G)-poly(C), releasing nearly 50% of the label in the added substrate as acid-soluble material. In contrast, the enzyme was much less active with poly(A)-poly(U) as substrate and was completely inactive with the single-stranded RNAs, poly(A) and poly(C) (data not shown). It is not surprising that the enzyme did not completely solubilize the poly(G)-poly(C), since such substrates are known to contain single-stranded regions although they elute in the double-stranded RNA fraction from cellulose columns (14). We conclude that the absB locus encodes a double strand-specific endoribonuclease, an RNase III, and we thus refer to the absB gene product hereafter as RNase IIIS (S for Streptomyces).



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  		FIGURE 1.
SDS-PAGE (12.5% gel) of fractions from the purification of AbsB (RNase IIIS). Lane 1, size standards with molecular weights as indicated, in thousands; lane 2, extract of uninduced cells bearing pJSE1811 (50 µg); lane 3, extract of induced cells (75 µg); lane 4, pooled fractions from the Talon column (0.9 µg); lane 5, dialyzed and concentrated AbsB (3.8 µg).

 



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  		FIGURE 2.
Activity of RNase IIIS (AbsB) on synthetic double-stranded substrates. Assays were performed as described under "Experimental Procedures," and results are expressed as a percentage of the acid-insoluble counts in each assay tube that were converted to an acid-soluble form by the action of RNase IIIS.

 
Organization of the rpsO-pnp Intergenic Region of S. coelicolor and Synthesis of the Read-through Transcript Substrate—A PCR fragment containing the S. coelicolor rpsO-pnp intergenic region was synthesized using primers rps1 and +16R (TABLE ONE) and cloned in pCR2.1-TOPO (Invitrogen), downstream of the T7 promoter borne by the vector. The sequence of the PCR fragment and associated vector sequences is shown in Fig. 3. The fragment includes 153 bases from the 3'-end of rpsO, the rpsO-pnp intergenic region, and 26 bases from the pnp open reading frame. The sequences thought to form the rpsO terminator are shown in the figure as are the sequences that we believe form the stem-loop structure upstream of pnp. The –10 and –35 regions of S. antibioticus Ppnp are identical to sequences found in the S. coelicolor intergenic region (Fig. 3) (9), and we posit that those sequences represent the intergenic promoter in S. coelicolor. The transcription start point specified by that promoter would be the T marked by an asterisk in Fig. 3.



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  		FIGURE 3.
Sequence of the insert of pJSE5600. The sequence of pCR2.1 TOPO, included in pJSE5600 is shown in lowercase type, and sequence from the rpsO-pnp intergenic region is in uppercase. The primers used to synthesize the rpsO-pnp fragment (rps1 and +16R), the primer used to map the RNase IIIS cleavage site in the 5'-vector sequence (Px4), and the primers used to verify the 3'-end of the insert (TOPO R1 and R2), are shown in boldface type. The rpsO terminator and the intergenic hairpin upstream of the pnp open reading frame are shown in italics and indicated by single and double arrows, respectively. The rpsO stop codon and the pnp start codon are double underlined. The putative –10 and –35 regions of the intergenic promoter, Ppnp, are underlined, and the transcription start site is indicated in boldface type with an asterisk. The BamHI site used to linearize the plasmid is boxed, as is the T7 promoter.

 
To synthesize a substrate for RNase IIIS, the pCR2.1-TOPO derivative described above, designated pJSE5600, was linearized with BamHI (site shown in Fig. 3) and transcribed with T7 RNA polymerase. For the experiments shown below, the transcript was collected by phenol extraction of reaction mixtures and ethanol precipitation. Essentially identical results were obtained with transcripts prepared by this method and with transcripts purified by polyacrylamide gel electrophoresis and elution prior to RNase IIIS digestion. It should be noted that the rpsO-pnp transcript studied here contains 72 and 42 bases at the 5'- and 3'-ends, respectively, derived from the pCR2.1-TOPO vector sequence (sequence in lowercase in Fig. 3). Secondary structure modeling studies indicated that these additional bases did not interfere with the formation of the rpsO terminator or the stem-loop structure, which we expected to be the target for RNase IIIS cleavage.

Cleavage of the rpsO-pnp Transcript by RNase IIIS—Transcripts of the rpsO-pnp intergenic region, labeled internally with [32P]CTP, were utilized as substrates for RNase IIIS as described under "Experimental Procedures." Reaction mixtures contained 3 µg of transcript and varying amounts of purified RNase IIIS. Reaction products were separated by electrophoresis on polyacrylamide gels in the presence of 7 M urea. As shown in Fig. 4, cleavage of the rpsO-pnp transcript produced two major and two minor fragments, at low ratios of enzyme to substrate (Fig. 4, lane 4). The measured sizes of the two major fragments, utilizing the RNA size standards shown in Fig. 4 as references, are ~300 bases (fragment 1) and 170 bases (fragment 2). We believe that fragments 1 and 2 are the products of a single cleavage event (see below) and that their actual sizes are 373 and 200 bases, respectively. We argue that these two fragments migrate anomalously, even in the presence of 7 M urea, because of the persistence of secondary structure after RNase IIIS cleavage (20). At higher ratios of enzyme to substrate the intensities of fragments 1 and 2 decrease, whereas those of fragments 3 and 4 increase (Fig. 4, lanes 5 and 6). These results are consistent with the hypothesis that after the initial RNase IIIS cleavage that produces fragments 1 and 2, fragment 1 is cleaved further to produce fragment 3, and fragment 2 is cleaved further to produce fragment 4.

To test this hypothesis, the rpsO-pnp transcript was labeled at either its 3'-or5'-end as described under "Experimental Procedures," and the resulting products were used as substrates for RNase IIIS. As shown in Fig. 4, lanes 8–10, treatment of the 3'-end-labeled transcript with increasing amounts of RNase IIIS converted fragment 2 to fragment 4, as predicted. Treatment of the 5'-end-labeled transcript with increasing amounts RNase IIIS led to the initial appearance of fragment 1 followed by the disappearance of label from that migration position (Fig. 4, lanes 11–14). No labeled fragment 3 was observed with 5'-end-labeled transcript as the substrate. We postulate that, following the initial cleavage, fragment 1 is further processed at its 5'-end, resulting in the loss of the 5'-end label as demonstrated in Fig. 4. Additional evidence supporting this conclusion will be presented below.



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  		FIGURE 4.
Gel electrophoresis of RNase IIIS digests of the rpsO-pnp transcript. Conditions were as described under "Experimental Procedures." Lanes 1 and 2, Ambion Century Plus size standards with sizes given in bases; lane 3, undigested rpsO-pnp transcript; lanes 4–6, digests of the transcript with 50, 100, and 200 ng of RNase IIIS, respectively; lane 7, undigested 3'-end-labeled rpsO-pnp transcript; lanes 8–10, digest of the 3'-end-labeled transcript with 5, 10, and 25 ng of RNase IIIS, respectively; lane 11, undigested 5'-end-labeled rpsO-pnp transcript; lanes 12–14, digest of the 5'-end-labeled transcript with 10, 25, and 50 ng of RNase IIIS, respectively. The arrow indicates a fragment believed to result from processing of fragment 4 at its 3'-end.

 



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  		FIGURE 5.
Primer extension analysis of the products of RNase III digestion of the rpsO-pnp transcript. Lanes 1–4, DNA sequencing ladder generated using primer +16R, in the order T, G, C, A; lane 5, primer extension of undigested rpsO-pnp transcript; lanes 6–8, primer extension of transcript following digestion with 50, 100, and 200 ng of RNase IIIS. Conditions for RNase IIIS digestion and primer extension were as described under "Experimental Procedures."

 



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  		FIGURE 6.
Mfold model for the hairpin in the rpsO-pnp intergenic region of S. coelicolor. The primary and secondary cleavage sites in the rpsO-pnp transcript, identified by primer extension, are indicated by arrows. The –10 region of Ppnp, which is a part of one of the stems of the hairpin, is also shown in boldface type. The sequence of the non-coding DNA strand is shown.

 
Primer Extension Identifies the RNase IIIS Cleavage Sites in the rpsO-pnp Transcript—In order to identify the RNase III cleavage sites in the rpsO-pnp transcript, we performed primer extension analyses of intact and cleaved transcripts, using primer +16R. Results of this analysis are shown in Fig. 5. The figure shows the DNA sequencing ladder generated by +16R and the extension products derived from transcripts treated with 0–200 ng of RNase IIIS. No extension product corresponding to a cleavage site was observed in the absence of RNase IIIS, as expected, although we did observe a higher molecular weight product, not shown in Fig. 5, that may represent the full-length transcript. Digestion of the rpsO-pnp transcript with RNase IIIS, followed by primer extension revealed two bands. The major band, band 1, identifies a cleavage site at the T residue (a U residue in the transcript) putatively identified as the start site for transcription from Ppnp (see Fig. 3). The intensity of the major band and the fact that it appears at relatively low ratios of RNase IIIS to substrate suggest that this band results from the primary cleavage event in the processing of the rpsO-pnp read-through transcript by RNase IIIS. The minor band shown in Fig. 5, band 2, is of lower intensity, and the amount of that band increases at higher ratios of enzyme to substrate. This band identifies a cleavage site 32 bases 3' of the primary cleavage site. The secondary structure model for the intergenic stem loop in S. coelicolor is shown in Fig. 6, and the primary and secondary cleavage sites are indicated. It is apparent that the primary cleavage occurs on the 5'-side of the stem-loop in one strand of a symmetric internal loop. The secondary cleavage occurs on the 3'-side of the stem-loop one base downstream of the 3'-end of the internal loop. These results are consistent with a model in which the initial RNase IIIS cleavage of the rpsO-pnp transcript produces fragments 1 and 2 of Fig. 4, and the secondary cleavage converts fragment 2 to fragment 4.

Additional primer extension experiments were performed as controls and to identify other RNase IIIS cleavage sites in the substrate. As controls, we performed primer extensions using primers TOPO R1 and TOPO R2, complementary to sequences at the 3'-end and near the middle of the pCR2.1-TOPO 3'-sequence shown in Fig. 3. These experiments confirmed that the rpsO-pnp transcript did terminate at the BamHI site shown in Fig. 3 (data not shown). We also performed primer extensions using primer Px4 to identify the cleavage site responsible for the conversion of fragment 1 to fragment 3 (Fig. 4). These experiments revealed that fragment 3 is produced by a cleavage that removes 40 bases from fragment 1. The cleavage site in question is located in the 5'-pCR2.1-TOPO sequence (data not shown), not in the rpsO-pnp region. Thus, cleavage at this site is not relevant to the processing of the rpsO-pnp intergenic region in Streptomyces, although it should be noted that secondary structure modeling does predict the presence of double-stranded structures in this region. Primer extension with Px4 also demonstrated that the rpsO-pnp transcript initiated at the residue numbered 1 in Fig. 3.

Kinetic Analysis of RNase IIIS Cleavage of the rpsO-pnp Transcript—The identification of the RNase IIIS cleavage sites in the model transcript allowed us to develop a kinetic assay utilizing that substrate to determine kinetic parameters for the enzyme. In these experiments, we measured the initial cleavage reaction only, viz. the production of fragments 1 and 2. This was accomplished by utilizing an enzyme concentration (79.1 nM) and a reaction time (2.5 min) that minimized subsequent processing of the substrate. Substrate concentrations from 0.27 to 2.7 µM were used in these analyses, and autoradiograms of the gels showed essentially no production of fragments 3 and 4 under the assay conditions just described.

A Lineweaver-Burk plot obtained for the production of fragment 1 by RNase IIIS cleavage is shown in Fig. 7, and kinetic parameters obtained by regression analysis of the plot are presented in TABLE TWO, along with parameters for cleavage of the T7R1.1 RNA substrate by E. coli RNase III. The Km value for RNase IIIS is at least an order of magnitude higher than the values reported for RNase III with T7 R1.1, and kcat/Km is 2–4-fold lower (1, 21, 22).


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  		TABLE TWO
Kinetic parameters for RNase IIIS and E. coli RNase III

Kinetic parameters were calculated from Fig. 7. Column 3 shows the temperature of the incubation and the source of monovalent ions. The kcat value for RNase IIIS was calculated using the molecular weight of the monomeric form of the enzyme. We do not yet know if the native form of RNase IIIS is a dimer.

 



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  		FIGURE 7.
Lineweaver-Burk plot of kinetic data. Kinetic assays were performed as described under "Experimental Procedures" under conditions that allowed only the initial cleavage of the rpsO-pnp substrate. The amounts of fragment 1 produced were determined and used to calculate initial velocities. Velocities are expressed as nmol min–1, and substrate concentrations are in µM. Data in the table are averages of duplicates ± S.E. Kinetic parameters were calculated from the intercepts of the plot.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The results presented above demonstrate that the product of the absB locus of S. coelicolor (11) is an RNase III capable of digesting synthetic double-stranded (but not single-stranded) RNA substrates and cleaving model transcripts at specific sites. A scheme for the action of RNase IIIS on the rpsO-pnp transcript derived from pJSE5600 is shown in Fig. 8. The transcript is cleaved initially at the U residue identified in Fig. 6 to produce fragments 1 and 2. Fragment 1 is subsequently cleaved, at higher ratios of enzyme to substrate and at a site located in the pCR2.1-TOPO sequence that is included in the transcript, to produce fragment 3. Fragment 2 is subsequently cleaved, again at higher ratios of RNase IIIS to transcript, to produce fragment 4. Careful inspection of Fig. 4 indicates that fragment 4 may be processed further (note the fragment identified by the arrow in Fig. 4), but we have not yet identified the cleavage sites responsible for this processing. We do not see this fragment in digests of 3'-end-labeled transcript (Fig. 4, lanes 8–10); thus, the cleavage site(s) must be situated at the 3'-end of fragment 4. As is the case with the cleavage site(s) involved in the conversion of fragment 1 to fragment 3, we suspect that the further processing of fragment 4 involves cleavage in the pCR2.1-TOPO sequence and that these reactions are not relevant to the processing of rpsO-pnp.

The kinetic parameters we obtained for the primary cleavage of the rpsO-pnp model transcript by RNase IIIS differ significantly from those reported for RNase III cleavage of T7 R1.1 RNA. However, our studies were obviously performed with a different enzyme, a different substrate, and under different reaction conditions than the RNase III studies. Indeed, as shown in TABLE TWO, different kinetic parameters have been obtained by different laboratories utilizing the T7 R1.1 substrate (1, 21, 22). The data of Fig. 7 and TABLE TWO clearly show that our assay can be used to determine kinetic parameters, and we will be able to use the kinetic assay to examine the cleavage of other streptomycete RNAs by RNase IIIS.

Our data show that, as is the case in E. coli, an intergenic stem-loop structure plays an important role in the processing of the rpsO-pnp transcript in S. coelicolor. Unlike the situation in E. coli, where both the read-through transcript and the transcript from Ppnp are processed at identical sites in the stem-loop (6), only the read-through transcript can form a stem-loop in S. coelicolor; no such structure can be formed by the transcript from Ppnp, since transcription from that promoter eliminates some 20 bases required for the formation of the 5'-stem of the hairpin. As indicated above, computer modeling suggests that the transcript from Ppnp forms a cloverleaf structure with several single stranded regions (10). Since we have identified several putative processing sites in those regions in our in vivo analyses, we speculate that in Streptomyces the transcript from Ppnp is processed primarily by a single strand-specific endonuclease, like RNase E, whereas the read-through transcript is processed primarily by RNase IIIS (10). The data reported here are consistent with this hypothesis.



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  		FIGURE 8.
Model for the cleavage of the rpsO-pnp transcript by RNase IIIS to produce the major bands shown in Fig. 4. The top line shows a schematic diagram of the transcript obtained from pJSE5600 with the vector sequences indicated by the thick lines. The primary cleavage produces fragments 1 and 2, the secondary cleavage produces fragment 4, and the cleavage at the 5'-end of the vector sequence produces fragment 3. The 40-base and 32-base fragments are not shown in Fig. 4, because they would have run off the end of the gel.

 
Another significant difference between E. coli and Streptomyces as regards rpsO-pnp transcript processing is the site of RNase III cleavage in the two systems. In E. coli, RNase III cleaves within a base-paired stem situated just below an internal loop in the hairpin (6, 7). In contrast, the primary cleavage site in S. coelicolor occurs within an internal loop, and the secondary cleavage occurs just 3'of the end of that loop (Fig. 6). These cleavage sites are similar to those reported for the processing of bacteriophage T7 mRNAs. Calin-Jageman and Nicholson (23) have studied the cleavage of primary transcription products from the T7 early genetic region by RNase III and have shown that several of the processing signals involve primary cleavage at a single site in an internal loop with secondary cleavage on the opposite side of that loop. In the T7 transcripts, the internal loops are asymmetric, with differing numbers of unpaired bases on the 5'- and 3'-sides of the loop (e.g. the [4/5] asymmetry). However, Calin-Jageman and Nicholson (23) did synthesize substrates with symmetric [4/4] internal loops and showed that those substrates could be cleaved by RNase III. They observed cleavage of both sides of the internal [4/4] loops in a fashion that was independent of the actual sequence of bases in those loops (23). The model for the stem-loop structure from the S. coelicolor rpsO-pnp transcript (Fig. 6) postulates the formation of a [4/4] internal loop, and our data show clearly that, as is the case with similar loops in the T7 transcripts, RNase IIIS cleaves both strands of the internal loop. Our results are consistent with the conclusion of Calin-Jageman and Nicholson (23) that it is the features of the internal loop that determine the specificity of cleavage of such loops by RNase III.

In E. coli, RNase III cleavage of the pnp transcripts is required for autoregulation of pnp expression (7–9). We do not know whether pnp expression is autoregulated in Streptomyces, but we have some evidence suggesting that it is. We overexpressed pnp using an inducible promoter in S. antibioticus from two plasmid constructs, pJSE340, containing the pnp open reading frame, the entire rpsO-pnp intergenic region, and some upstream sequence, and pJSE343, containing only the pnp open reading frame (24). Induction of pnp expression in the strain bearing pJSE340 led to an initial 2.5–3-fold increase in PNPase activity followed by a gradual return to wild type levels. With the strain bearing pJSE343, on the other hand, PNPase-specific activity increased steadily for several days after the induction of pnp expression (24). It is thus tempting to speculate that pnp expression is autoregulated in Streptomyces and that autoregulation requires sequences in the rpsO-pnp intergenic region, sequences that are present in pJSE340 but absent from pJSE343.

As indicated above the, absB locus was identified by virtue of its function in the global control of antibiotic production in S. coelicolor (11). Mutations in absB abolish production of all four antibiotics normally synthesized by S. coelicolor (11, 12, 19). Since we have shown that absB is an RNase III, it seems quite likely that the biochemical activity of the enzyme is responsible for its regulation of antibiotic production. There are two obvious mechanisms to explain the role of RNase IIIS in the regulation of antibiotic production in S. coelicolor. RNase IIIS might be required to process the transcript for an activator of antibiotic production. In the absB mutant, that activator would not be produced, at least not in its active form. Alternatively, RNase IIIS might be required to degrade the transcript for a repressor of antibiotic synthesis. In the absB mutant, that transcript and its protein product would persist. The actual situation is likely to be more complex than these simple possibilities suggest. We plan to begin microarray studies to identify the putative regulatory targets for absB. The availability of a kinetic assay for RNase IIIS will make it possible to compare the properties of the substrates identified by the microarray analysis with each other and with the rpsO-pnp transcript described here.


   FOOTNOTES
 
* This work was supported by National Science Foundation Grant MCB-0133520. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel.: 404-727-0712; Fax: 404-727-2880. E-mail: george.h.jones{at}emory.edu.

2 The abbreviation used is: PNPase, polynucleotide phosphorylase. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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