Overexpression, purification, and properties of Escherichia coli ribonuclease II.

Ribonuclease II (RNase II) is a major exonuclease in Escherichia coli that hydrolyzes single-stranded polyribonucleotides processively in the 3′ to 5′ direction. To understand the role of RNase II in the decay of messenger RNA, a strain overexpressing the rnb gene was constructed. Induction resulted in a 300-fold increase in RNase II activity in crude extracts prepared from the overexpressing strain compared to that of a non-overexpressing strain. The recombinant polypeptide (Rnb) was purified to apparent homogeneity in a rapid, simple procedure using conventional chromatographic techniques and/or fast protein liquid chromatography to a final specific activity of 4,100 units/mg. Additionally, a truncated Rnb polypeptide was purified, solubilized, and successfully renatured from inclusion bodies. The recombinant Rnb polypeptide was active against both [3H]poly(A) as well as a novel (synthetic partial duplex) RNA substrate. The data show that the Rnb polypeptide can disengage from its substrate upon stalling at a region of secondary structure and reassociate with a new free 3′-end. The stalled substrate formed by the dissociation event cannot compete for the Rnb polypeptide, demonstrating that duplexed RNAs lacking 10 protruding unpaired nucleotides are not substrates for RNase II. In addition, RNA that has been previously trimmed back to a region of secondary structure with purified Rnb polypeptide is not a substrate for polynucleotide phosphorylase-like activity in crude extracts. The implications for mRNA degradation and the proposed role for RNase II as a repressor of degradation are discussed.

Because the rate of synthesis of any given protein is directly proportional to the concentration of its message, regulating the balance between mRNA decay and its synthesis is an important aspect of gene expression. In Escherichia coli, it is widely accepted that mRNA decay is initiated by a series of endonucleolytic cleavages catalyzed by RNase E (1)(2)(3) or occasionally by RNase III (4,5) followed by processive exonucleolytic degradation of the message to oligo-and mononucleotides (1)(2)(3). Two 3Ј-exonucleases have been implicated in this process: ribonuclease II (RNase II) 1 and polynucleotide phosphorylase (PNPase) (6). RNase II, which is responsible for the majority of the exonucleolytic activity in E. coli extracts (7), hydrolyzes RNA to release 5Ј-mononucleotides (8), while PNPase phospho-rylyzes RNA to mononucleoside diphosphates (9). Although RNase II activity was first described over three decades ago (10,11) and purified from whole cells several years later (12)(13)(14), details of its role in mRNA degradation are still poorly understood.
RNA structure, known to be an important determinant of mRNA stability, can protect upstream sequences from digestion by the 3Ј-exonucleases. The Rho-independent terminator sequence (trp t) of the tryptophan operon (15) and the intergenic (malE-malF) REP sequence of the maltose operon (16) are classic examples of secondary structures that protect upstream RNA from 3Ј-exonucleolytic degradation both in vitro and in vivo. These investigations implied that the observed protection by 3Ј-stem-loop structures was the result of an impediment to the processive activities of RNase II and, to a lesser extent, of PNPase (15)(16)(17). Recent observations of the decay of RNA-OUT, the antisense RNA that regulates Tn10/ IS10 transposition, demonstrate that the higher the thermal stability of the RNA structure, the larger the barrier to degradation by RNase II (18). Degradation by PNPase is much less affected by the relative stability of the RNA-OUT structure (18). Interestingly, RNA-OUT appears to be stabilized approximately 3-fold against PNPase attack by RNase II (18). In addition, the rpsO mRNA is also stabilized significantly by the presence of RNase II (19). Although the mechanism by which RNase II shelters upstream sequences from further exonucleolytic attack is not understood, the observed protection was attributed to the formation of a stable RNase II-RNA complex, which sequesters the 3Ј-end of the transcript (18,19).
As part of the investigation of the functional and biophysical properties of RNase II and its role in the overall decay of mRNA, we have overexpressed RNase II and developed a rapid and simple purification of the enzyme free of other nucleases. The purified enzyme was used to investigate the mechanisms by which stem-loop structures impede exonucleases and the ability of RNase II to act as a repressor of PNPase activity.

EXPERIMENTAL PROCEDURES
Bacterial Strains and Plasmids-The E. coli strain 18 -11 (rna Ϫ , rnb Ϫ , rnd Ϫ , rbn Ϫ , rnt Ϫ ) (20) was obtained from Dr. M. P. Deutscher (University of Connecticut Health Center, Farmington), while the strain CF881 F ⌬lac argA trp recB1009 ⌬(xthA-pnc) ⌬rna was obtained from Dr. M. Cashel (National Institutes of Health). The vector pET-11 and its host stain BL21(DE3) (21) were obtained from Novagen. The plasmid pRP40 (22) was obtained from Dr. N. Sonenberg (McGill University, Montreal). The following oligonucleotide primers were synthesized based on the previously published rnb sequence (23): fP1 (5Ј-GCGAGGATCCAGGAGGTGACAATTATGTTTCAGGACAAC) and rP1 (5Ј-GCGAGGATCCTTTCCATGCGGACTTCGGCATTA). An additional reverse primer rP2 (5Ј-GCGAGGATCCATCGACGGTCAGACTCATCA-TCA) was constructed based on the partial DNA sequences of pRZA17 and pRZA18 obtained from Dr. C. M. Arraiano (Centro de Tecnologia Química e Biológica, University of Lisbon, Portugal) which contain the 3Ј-untranslated region of the rnb gene. The predicted coding sequence of the rnb gene of E. coli was amplified from genomic DNA of strain MV1190 by the polymerase chain reaction. The products were cleaved with BamHI and ligated into the unique BamHI site of pET-11. The orientation of the 2.4-(fP1-rP2) and 1.9-kilobase pair (fP1-rP1) BamHI fragments in the recombinant plasmids was verified by restriction mapping and DNA sequencing of the entire rnb gene. The resulting plasmids, pGC100 and pGC101, were used to transform BL21(DE3) to yield strains GC100 and GC101, respectively.
RNase II Assays-The 92-nucleotide (nt) partial duplex RNA substrate, which we call t40B (previously called RNA I) (22), was generated from the plasmid pRP40 linearized with the restriction enzyme BamHI. Synthesis of uniformly labeled t40B was directed from an SP6 promoter in the presence of [␣-32 P]CTP as described previously (24). Assays for RNase II activity were assembled in a 70-l reaction volume containing 10 pmol of labeled t40B in a reaction buffer containing 17 mM HEPES⅐NaOH, pH 7.5, 0.5 mM MgAc 2 , 100 mM KCl, 2 mM DTT, 5% glycerol, and 10 g/ml acetylated bovine serum albumin (New England Biolabs). Protein was added last to the final concentration specified in the figure legends, and incubations were performed at 37°C. Samples were withdrawn at various times and quenched in 3 volumes of loading buffer containing 90% deionized formamide, 22 mM Tris, 22 mM boric acid, 0.5 mM EDTA, 0.1% xylene cyanol FF, and 0.1% bromphenol blue. The products were resolved by electrophoresis on 10% polyacrylamide gels containing 8 M urea and visualized by autoradiography or with a Molecular Dynamics PhosphorImager system. Activity was also determined by release of acid-soluble radioactivity from [ 3 H]poly(A) (25). 1 unit of RNase II activity is defined as the release of 1 mol of AMP/h.
Preparation of Crude Extracts-Cultures of CF881 and 18 -11 grown in 1 liter of rich medium (21) to late logarithmic phase were harvested by centrifugation and frozen at Ϫ70°C until use. The thawed cells were resuspended in 3 volumes of buffer A (60 mM Tris⅐HCl, pH 7.5, 10 mM MgCl 2 , 60 mM NH 4 Cl, 0.05 mM EDTA, 1 mM DTT) and ruptured by passage through an Aminco French pressure cell at 15,000 p.s.i. The cell lysate was centrifuged at 30,000 ϫ g for 30 min in a Beckman JA-20 rotor at 4°C. The supernatant (S-30) was then centrifuged at 150,000 ϫ g in a Beckman Ti70.1 rotor for 2 h at 4°C. The supernatants, S-30 and S-150, were the source of crude extracts for subsequent experiments.
Preparation of RNase II (Rnb) from an Overexpressing Strain-Cultures of GC100 were grown in a rich medium (21) at 30°C to early logarithmic phase and induced with 0.4 mM isopropyl ␤-thiogalactopyranoside (IPTG) for 5 h. The cultures were chilled, and the cells were harvested by centrifugation at 4,000 ϫ g for 10 min. All subsequent procedures were performed at 4°C. Cell pellets were resuspended in 20 -25 ml of buffer B containing 50 mM HEPES⅐NaOH, pH 7.5, 500 mM NaCl, 1 mM MgCl 2 , 0.1 mM EDTA, 5 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, 0.8 g/ml leupeptin, and 2 g/ml aprotinin. The cells were ruptured by passage through an Aminco French pressure cell at 15,000 p.s.i. The lysate was centrifuged at 30,000 ϫ g for 60 min in a Beckman JA-20 rotor to pellet unbroken cells and insoluble material. Approximately 60 mg of the S-30 was loaded onto a column of Affi-Gel blue (Bio-Rad) (1.25 ϫ 21.5 cm) previously equilibrated with 3 column volumes of buffer C (25 mM HEPES⅐NaOH, pH 7.5, 5% glycerol, 2 mM DTT, 1 mM MgCl 2 , 0.1 mM EDTA) containing 500 mM NaCl. The column was washed with 3-5 column volumes of this buffer at a flow rate of 8.3 ml/h (6.75 cm/h) driven by a P1 peristaltic pump (Pharmacia Biotech Inc.). The Rnb polypeptide was eluted with 5 column volumes of buffer C containing 3 M NaCl. The eluent was pumped directly onto a column of hydroxylapatite (Bio-Rad) (0.75 ϫ 8.5 cm) at a flow rate of 6.7 ml/h (15 cm/h). After washing with 5 column volumes of buffer C containing 1 mM sodium phosphate, pH 7.5, the Rnb polypeptide was eluted with a 50-ml gradient of sodium phosphate, pH 7.5 (1-250 mM), in buffer C at a concentration of 75 mM sodium phosphate. Fractions containing the Rnb polypeptide were divided into pool A or pool B based on the contaminants present in the fractions. A portion of pool A was loaded onto a column of Affi-Gel heparin (Bio-Rad) (0.75 ϫ 8.0 cm). The column was washed with 3-5 column volumes of buffer C at a flow rate of 7.2 ml/h (16 cm/h). The Rnb polypeptide was eluted from the column with a 50-ml gradient of NaCl (0 -400 mM) in buffer C at a concentration of 130 -140 mM NaCl. Alternatively, chromatography (FPLC) of pool A on a Resource Q column (Pharmacia) was substituted for the Affi-Gel heparin step. After loading the sample and washing it with 5 column volumes of buffer C containing 150 mM NaCl, the Rnb polypeptide was eluted from this resin with a 50-ml gradient of NaCl (100 -400 mM) in buffer C at a concentration of 220 mM NaCl. The presence of the Rnb polypeptide in various fractions was monitored qualitatively by polyacrylamide gel electrophoresis and quantitatively by enzyme assay (see above). The pooled fractions obtained from heparin-agarose chromatography were the source of purified Rnb polypeptide in all subsequent experiments.
UV Photocross-linking-Assay mixtures were prepared as described above with 160 fmol of t40B substrate. After incubation on ice for 2-5 min, the sample was subjected to a single 2-6-ns pulse (40 -50 mJ) with a 266-nm UV laser (Spectra Physics) as described previously (26). The sample was then incubated with 5 g of RNase A and 5 units of RNase T1 at 37°C for 45 min to remove excess RNA. Each digested sample was boiled in an equal volume of SDS sample buffer and separated electrophoretically on a 15% SDS-polyacrylamide gel. The cross-linked proteins were visualized by autoradiography.

Exonucleolytic Activity in Crude Extracts From E. coli-A
partially duplexed RNA substrate ( Fig. 1a) was used to assay extracts generated from various E. coli strains for putative RNA helicase activities. Instead of detecting an activity that could unwind the duplexed RNA to monomers, we observed the partial degradation of the synthetic substrate in extracts that are wild type for RNase II activity but not in extracts deficient for a number of exonucleases including RNase II (Fig. 1, compare b and c). Complete conversion of the 92-nt substrate to a relatively stable 77-nt degradative intermediate was observed in crude extracts prepared from strain CF881 over a 60-min time course (Fig. 1b). The exact size of the product was determined on a sequencing gel (data not shown). In contrast, crude extracts prepared from strain 18 -11 were unable to digest the substrate (Fig. 1c). Several additional experiments were undertaken to confirm that the 77-nt degradation product (shown in Fig. 1a) corresponds to the product of RNase II stalling 9 nucleotides 3Ј to the double-stranded region of the substrate. First, the denatured 77-nt product retains a 5Ј-end label (data not shown). Second, the partial duplex substrate is resistant to digestion by the purified Ams/Rne/Hmp-1 polypeptide, the catalytic subunit of RNase E (27), under conditions where authentic substrates would be processed to completion (data not shown). Third, incubation of the 92-nt substrate under conditions where PNPase, the other major exonucleolytic activity in E. coli, would be active also generates a 77-nt product but only in the presence of 10 mM sodium phosphate ( Fig., 2a). In this case, however, the 77-nt product can be degraded further in prolonged incubations (data not shown). Moreover, extracts prepared from a strain containing the mutant pnp-7 allele, which largely lacks PNPase activity but does contain RNase II activity, also generate the 77-nt product in the presence or absence of phosphate (data not shown). Although contributions from other exonucleases cannot be excluded completely, the phosphate-independent formation of the 77-nt product is most consistent with RNase II activity. This was confirmed (see below) using purified recombinant RNase II.
Overexpression and Purification of RNase II (Rnb)-The predicted coding sequence of the rnb gene of E. coli was amplified by the polymerase chain reaction as described under "Experimental Procedures." All primers contained BamHI restriction sites, and fP1 also contains a Shine-Dalgarno sequence 5Ј to the rnb start codon such that the amplified product could be cloned into the unique BamHI site of pET-11 and subsequently overexpressed using the T7 RNA polymerase encoded by BL21(DE3) (21). The partial structures of plasmids containing all or part of the rnb gene are depicted in Fig. 3, b and c. Due to errors in the previously published rnb sequence, which predicted a stop codon at position 2078 (23), plasmid pGC101 ( Fig.  3c) contains most of the rnb coding sequence except for a deletion of 26 nucleotide residues, which is replaced by 66 nucleotide residues of vector-derived sequence at the 3Ј-end of the construct. Plasmid pGC100 (Fig. 3b) contains the entire predicted 1932-nucleotide residue open reading frame, the 3Јuntranslated region including the putative Rho-independent terminator, and approximately 400 nucleotide residues of intercistronic spacer under the control of the T7-lac promoteroperator region in pET-11 (21).
Upon induction of cultures of GC100 or GC101 with IPTG, the Rnb polypeptide was expressed to the extent that it represented the most abundant polypeptide in whole cell extracts and a significant fraction of the total cellular protein (Fig. 4,  lane 3). When assayed against poly(A), crude extracts (S-30) from strain GC100 displayed a specific activity of 1,184 units/mg (Table I), 300-fold higher than that obtained from crude extracts prepared from the haploid strain CF881 (specific activity ϭ 3.9 units/mg). An efficient method of purification was developed in part by exploiting several effective steps from previously published methods (12)(13)(14). The initial step relies on Cibacron blue-agarose chromatography to remove the bulk of the nucleic acids and contaminating proteins while the majority (Ͼ90%) of the Rnb polypeptide remains bound to the column. Considerable efficiency was gained by loading the 3 M NaCl eluate from this column directly onto a hydroxylapatite column. This proved to be an invaluable step in the purification method since concentration, desalting, and significant purification of the Rnb polypeptide could take place in a single step. The apparent loss of activity after hydroxylapatite chromatography (Table I) may have been due to the inhibition by Ca 2ϩ ions leached from the column at high ionic strength, as Ca 2ϩ has been reported to inhibit RNase II activity (10,11). Final purification of Rnb from most contaminants could be achieved by affinity chromatography on heparin-agarose or by ion exchange chromatography (FPLC). A sample of the purified Rnb polypeptide is shown in Fig. 4, lanes 7 and 8. Based upon Coomassie Blue or silver staining of overloaded polyacrylamide  gels, the preparation was judged to be about 95% pure with a few faint minor contaminating bands. The specific activity of the Rnb polypeptide purified to the end of the heparin-agarose step was determined to be 4,100 units/mg, which is nearly 2-fold greater than that reported by others for the enzyme purified from whole cells (11)(12)(13). However, the specific activity of this preparation is approximately 2.5-fold lower than the best reported purification (14). It is quite possible that not all of the overexpressed Rnb polypeptide is properly folded or fully active. Nonetheless, this method provides a more rapid and facile purification of RNase II with good yields and activity.
If recoveries from the heparin-agarose chromatography step in Table I are extrapolated to include all the material in pool A from the hydroxylapatite column, the overall yield is 29%. This apparent overall yield is low for two reasons. First, the activity in crude extracts represents the sum of activities of a number of endo-and exonucleases and overstates the activity of RNase II. Second, fractions were pooled to maximize purity rather than yield particularly after hydroxylapatite chromatography.
The addition of 22 amino acid residues, derived from the vector pET-11, to the C terminus of the truncated Rnb* polypeptide, resulted in the formation of insoluble inclusion bodies upon induction of cultures of GC101 with IPTG. The inclusion bodies were subsequently purified to near homogeneity by differential centrifugation in the presence of detergent. Authentic RNase II activity was recovered following solubilization, reduction, and refolding of the truncated Rnb* polypeptide from the inclusion bodies. Further purification of the renatured truncated Rnb* polypeptide from most contaminants could be achieved by ion exchange chromatography (FPLC). The truncated Rnb* polypeptide eluted from the Resource Q column over a broad range of NaCl concentrations likely reflecting the several different populations of misfolded and inactive polypeptides present in the preparation. RNase II activity eluted from the column as a sharp peak at a NaCl concentration of 220 mM. Although a significant amount of activity could be recovered from the inclusion bodies, the specific activity of this preparation was quite poor, 54 milliunits/mg, a small fraction of that obtained for the full-length Rnb polypeptide.
Properties of the Rnb Polypeptide-The purified Rnb polypeptide was active against the partial duplex t40B substrate in a manner similar to the activity originally detected in crude extracts from strain CF881 (Fig. 5a, lanes 2-6). Under conditions in which enzyme is limiting (molar ratio of substrate to enzyme 2300:1), the 3Ј-single-stranded tails are removed from the substrate during a 60-min incubation at 37°C to generate a degradative intermediate, which has been shortened by about 15 nucleotides. The appearance of the degradative product is linear for 30 min, after which the rate declines gradually (Fig. 5a, lanes 2-6). Thus, each enzyme molecule is turning over more than 30,000 times.
Digestion of the t40B transcript is complete after a 60-min incubation with 2.0 milliunits of RNase II activity (Fig. 5a, lane  7). Approximately 20% of the substrate is resistant to degradation by the Rnb polypeptide even after addition of 200 milliunits of fresh enzyme (data not shown). A fraction of the substrate appears to form concatemers and as a result does not have free 3Ј-ends accessible to the enzyme. Interestingly, digestion of t40B for 60 min at 37°C with 4 units of enzyme resulted in a further shortened (73 nt) but stable degradation intermediate depicted by the arrowhead (Fig. 5a, lane 8). This experiment suggests that at high concentrations, the Rnb polypeptide can remove three to four additional unpaired residues in the t40B duplex remaining from a previous round of digestion.
Several previously published reports have suggested that RNase II, of varying degrees of purity, is readily inactivated by heat (10 -13, 28). We have tested the purified Rnb polypeptide and found that the recombinant enzyme is also susceptible to thermal inactivation (Fig. 5b). A comparison of Fig. 5b, lanes 2 and 3, shows that less than 1% of the activity remains after a 5-min incubation of the Rnb polypeptide in the absence of substrate at 37°C. Interestingly, the enzyme is stabilized in the presence of substrate and can remain active up to 60 min at 37°C (Fig. 5a). Activity can also be stabilized by the addition of substrate to Rnb polypeptide, which has been partially inactivated by a brief incubation in buffer at 37°C. Once activity has been lost to thermal inactivation, however, it cannot be regained upon addition of substrate (data not shown). The 77-nt product also stabilized the enzyme against heating. Rnb polypeptide was incubated in the presence of 1.5 pmol of partially digested t40B for 5 min at 37°C prior to incubation with full-length t40B transcript. The 77-nt product not only protected the Rnb polypeptide against thermal inactivation but also appeared to stimulate the activity of the enzyme for the full-length substrate by approximately 2-fold (data not shown). The apparent stimulation may be attributable to a decreased rate of thermal inactivation. Taken together, the data demonstrate that the enzyme can be stabilized by both substrate and product. In contrast, both a single-stranded DNA oligonucleotide (33-mer) and double-stranded plasmid DNA inhibited the activity of RNase II but were unable to provide significant protection from heating (data not shown) unlike oligonucleotides of deoxy(C) 27 , which can reduce the rate of thermal inactivation (28).
Stabilization of RNase II by the digested t40B transcript  implies that in the absence of any free 3Ј-single-stranded ends, the Rnb polypeptide can bind RNA even if it is not a substrate. To test this hypothesis, t40B was incubated briefly with a large excess of Rnb polypeptide, sufficient to digest it to 73 nt, and then subjected to UV photocross-linking. Fig. 6, lane 1, shows labeling of a band of 70 kDa, the size expected for the Rnb polypeptide. In addition, there is label associated with a band of 14 kDa, which we believe to be RNase A. The Rnb polypeptide is, therefore, able to bind its product (Fig. 6, lane 1) in the absence of any other proteins or cofactors. A 70-kDa protein, corresponding to the molecular mass of RNase II, was also labeled in crude extracts prepared from strain CF881 (Fig. 6,  lane 3). All bands were sensitive to proteinase K treatment (Fig. 6, lanes 2 and 4). A comparison of Fig. 6, lanes 1 and 3, also demonstrates that UV cross-linking can provide an important assessment of the purity of the enzyme preparation in light of the affinity chromatography techniques utilized in the purification. Since there are a large number of RNA binding proteins in crude extracts prepared from E. coli that have a significant affinity for the t40B transcript, the presence of even a small percentage these contaminants would be readily detected in the purified material (Fig. 6, compare lane 3 to lane 1).
We have also tested whether the 77-nt product would inhibit the activity of the Rnb polypeptide in subsequent rounds of digestion. In the first experiment, the Rnb polypeptide (3.3 milliunits) was incubated with 25 pmol of unlabeled t40B (2.5fold molar excess over labeled t40B) for 2.5 min at 37°C prior to addition of labeled t40B. The kinetics of digestion of labeled t40B over a 60-min time course were identical to those in an incubation in which the same amount of enzyme was incubated directly with labeled t40B (data not shown). In the second experiment, the t40B transcript, which had been previously digested with Rnb polypeptide, extracted with phenol/chloroform, and ethanol precipitated, was used in a competition experiment. Equimolar amounts of digested t40B did not alter the kinetics of disappearance of the 92-nt substrate and thus were unable to compete effectively for the Rnb polypeptide (Fig. 7). Although the 77-nt product can protect the enzyme from thermal inactivation, it cannot inhibit its activity.
Recent observations have suggested that RNase II can protect "upstream" RNA sequences from PNPase attack through the formation of a stable RNA-RNase II complex (18,19). We have further investigated this hypothesis by incubating the 77-nt product, produced by the action of the Rnb polypeptide (see above), with crude extracts prepared from strain 18 -11 in the presence of 10 mM sodium phosphate. The data demonstrate that the 77-nt product is resistant to digestion by a PNPase-like activity (Fig. 2b, lanes 1-4). As discussed above, the 92-nt substrate is rapidly shortened to approximately 77 nt in the presence of phosphate over a 30-min time course of digestion (Fig. 2a, lanes 5-8).

DISCUSSION
The Mechanism of Action of RNase II on a Novel Substrate-We envisage that the action of RNase II on t40B can be described by the following sequential steps: 1) binding to a free 3Ј-end on the 92-nt substrate, 2) processive hydrolysis of 15 phosphodiester bonds, 3) stalling of the enzyme approximately 9 unpaired nucleotides from the 10-bp G-C-rich stem, 4) dissociation of the enzyme from the substrate, and (5) thermal inactivation of a fraction of the dissociated enzyme. The duration of each such cycle at steady state can be calculated from the apparent turnover number, which we estimate as 9 nt⅐s Ϫ1 based on a rate of 0.16 pmol of product formed per min at 4.3 fmol of enzyme. This yields a cycle time of 1.67 s, the time to remove 15 nucleotides from each 3Ј-end (15 nt/9 nt⅐s Ϫ1 ). The time actually required for hydrolysis of 15 phosphodiester bonds (step 2 in the cycle) is only 0.21 s, however, as the reported turnover number for RNase II acting on poly(A) is 70 nt⅐s Ϫ1 (28). If we assume that this turnover number also applies to the 15 residues removed from t40B and that no enzyme is lost to thermal inactivation (step 5), then steps 1, 3, and 4 account for 1.46 s (1.67 Ϫ 0.21 s) of each cycle. As a consequence, RNase II cannot remain bound to a substrate once processive hydrolysis has ceased any longer than 1.46 s. The latter represents a maximum value for step 3 in the proposed cycle, as binding (step 1), dissociation (step 4), and thermal inactivation (step 5) are not negligible.
The linear kinetics observed for the reaction demonstrate that RNase II stalls at regions of secondary structure, however briefly, but can disengage from the "stalled" substrate and reassociate with a new free 3Ј-end. This is substantiated by the demonstration that the Rnb polypeptide can cycle from an unlabeled to a labeled substrate. Our finding of dissociation from a substrate with 9 unpaired protruding nucleotides at the 3Ј-end of the 77-nt product is in good agreement with the 10 -15-nt digestion limit product obtained for RNase II acting FIG. 6. UV cross-linking of t40B to purified recombinant Rnb polypeptide and proteins in the S-150 fraction prepared from strain CF881. Labeled t40B was incubated with purified recombinant Rnb polypeptide (2.1 units, 50 g/ml) or with 10 g of an S-150 fraction prepared from strain CF881 (33 milliunits) at a concentration of 1 mg/ml, irradiated with UV, digested with ribonucleases, and then separated by SDS-PAGE as described under "Experimental Procedures." A duplicate sample was treated with proteinase K( ϩ ) (PROT K) prior to electrophoresis (lanes 2 and 4). Lanes 1 and 2, purified recombinant Rnb polypeptide; lanes 3 and 4, crude extract prepared from strain CF881.
FIG. 7. Competition between partially digested t40B and complete t40B. Rnb polypeptide (2.0 milliunits, 8.3 ng/ml) was incubated with intact t40B in the presence (᭜) or absence (f) of 10 pmol of t40B that had been previously digested to yield a 77-nucleotide product with the purified Rnb polypeptide. The products were resolved by gel electrophoresis, and the relative amounts of t40B were quantified with a PhosphorImager, expressed as picomoles of RNA remaining, and plotted as a function of time.
on homopolymers (28). Interestingly, RNase II can participate in the processing of some tRNAs in vitro by degrading long trailing sequences but must be able to dissociate from the precursor to allow final maturation of the tRNA by other processing exonucleases (29).
Two lines of evidence suggest that the Rnb polypeptide can also reassociate with the 77-nt product of digestion. First, at high concentrations of enzyme the Rnb polypeptide can remove three to four additional unpaired residues remaining from a previous round of digestion. Second, the Rnb polypeptide can bind its 73-77-nt product as evidenced by UV cross-linking and protection from thermal inactivation. Although partially digested t40B can bind to the Rnb polypeptide, it does not compete with the full-length substrate, indicating that the preferred substrate for RNase II has an extended free 3Ј-end. Moreover, the lack of competition by product implies that product binds to a site distinct from that of the substrate.
A Model for the Control of mRNA Degradation at the 3Ј-End-As discussed in the Introduction, 3Ј-stem-loop structures have been shown to protect upstream RNA sequences from digestion by 3Ј-exonucleases (15)(16)(17). The observed protection of upstream sequences was originally attributed to the impeding of the processive activities of RNase II or PNPase by RNA structure. Our results, however, demonstrate that the Rnb polypeptide loses its apparent processivity nine residues 3Ј to a region of strong RNA secondary structure, where it leaves the substrate rapidly and reassociates with a new free 3Ј-end. The data imply that the recently observed stabilization of the Tn10/ IS10 antisense RNA-OUT (18) and the stabilization of rpsO mRNA (19) by RNase II are probably due to the removal of the 3Ј-overhang rather than to the formation of a stable RNA-RNase II complex, which blocks access of PNPase to the 3Ј-end of mRNAs. However, it should be noted that dissociation and/or binding events could be retarded in vivo if free 3Ј-ends are limiting or if stem-loop binding proteins stabilize an RNase II-product complex (16,30,31). It has been suggested that mRNAs with an immediate 3Ј-stem-loop structure, analogous to the 77-nt product, are poor substrates for PNPase (32). Our data demonstrate that a PNPase-like activity in crude extracts can degrade the t40B substrate in a phosphate-dependent manner while the 77-nt product, produced by the action of the purified Rnb polypeptide, is not an efficient substrate. Conceivably, extension of the 3Ј-end by poly(A) polymerase (PcnB) (32)(33)(34)(35) could provide a necessary single-stranded platform for PNPase to overcome the apparent indirect inhibition by RNase II.
These observations suggest a possible model for the control of mRNA degradation at the 3Ј-end. As RNase II encounters a region of secondary structure, it stalls. If the structure is unstable, the enzyme may advance through the stem-loop in the 3Ј to 5Ј direction. However, if the structure is a stable REP sequence or a Rho-independent terminator, RNase II will dissociate from the transcript before the duplex opens. We propose that loss of the single-stranded 3Ј overhang, which reduces the affinity of RNase II for the stalled transcript, may also reduce the ability of the much larger PNPase to bind and degrade transcripts. It could also reduce the affinity of putative RNA helicases for such structures. Addition of a new 3Ј-end by PcnB followed by the action of PNPase, known to be less susceptible to RNA secondary structure than RNase II (15)(16)(17)(18)(19), would be required for the degradation of strong REP and terminator sequences. Thus, a competition between removal of a 3Ј-overhanging sequence by RNase II and extension-degradation by PcnB and PNPase, respectively, would develop at the 3Ј-end of extended RNA secondary structures and may account for the heterogeneity in the 3Ј-ends of oligoadenylated RNA I (33).
Utility of t40B as a Substrate for RNase II Activity-This partially duplexed RNA is an effective substrate for investigating the properties of RNase II and offers at least four significant advantages over assays previously utilized for detecting RNase II activity. First, the t40B transcript resembles natural mRNA substrates more closely than the homopolymeric substrates utilized in traditional assays as it contains both 3Јunpaired extensions of essentially random composition and a stable duplex mimicking stem-loop structures found in natural mRNAs. Second, the stalling of the enzyme at the duplexed region reflects the known behavior of RNase II on RNAs containing regions of extensive secondary structure (15)(16)(17)(18). Third, the formation of a stable degradative intermediate provides an internal control that distinguishes RNase II activity from single and double strand-specific endonucleases. Finally, the high specific activity of the synthetic transcript increases the sensitivity of the assay and allows for the detection of activity at low substrate concentrations (10 Ϫ10 -10 Ϫ11 ) closer to the physiological range.