A New Endoribonuclease from Escherichia coZi RIBONUCLEASE N*

A new ribonuclease called RNase N was isolated from Escherichia coli. It is a nonspecific endoribonuclease that can cleave rRNA, poly(U), and poly(C) to small oligonucleotides and 5'-mononucleotides. It requires monovalent cations and is inhibited by divalent cations. It is suggested that this enzyme plays a role in the decay of rRNA,under various starvation conditions and perhaps in the decay of mRNA.

A new ribonuclease called RNase N was isolated from Escherichia coli. It is a nonspecific endoribonuclease that can cleave rRNA, poly(U), and poly(C) to small oligonucleotides and 5'mononukleotides.
It requires monovalent cations and is inhibited by divalent cations. It is suggested that this enzyme plays a role in the decay of rRNA, under various starvation conditions and perhaps in the decay of mRNA.
In a series of studies on the turnover of ribosomes in Escherichia coli during deprivation of various nutrients essential for growth, it was concluded by Kaplan and Apirion (3,4) that a common mechanism operates during the turnover of rRNA under the various starvation conditions. Their in vivo studies implicated that the turnover process is initiated by an endonucleolytic attack on rRNA in ribosomal subunits. From some detailed analyses of the process (3) and a review of current hterature about ribonucleases in E. coli (5, 6) it could have been considered that the enzyme, RNase III, which degrades double stranded RNA (71, could have been this enzyme. However, when RNase III+ and RNase III-strains were compared, it was found that degradation of rRNA during carbon starvation was not reduced in RNase III-strains (8,9), thus, eliminating this possibility.
To understand the lacuna of the process, we started to search for a new ribonuclease(s) in cell extracts which could endonucleolytically attack rRNA, preferentially in ribosomes. We shall report here that at least one and perhaps two such enzymes are present in the E. coli cell. in Buffer F and dialyzed overnight against 100 volumes of the same buffer with two changes. Each fraction was tested for the degrading activity of RNA in the ribosome.

DEAE-Sephadez
Chromatography-DEAE-Sephadex A-50 was packed in a column (45 x 2.5 cm) at a flow rate of 35 ml/h and equilibrated with Buffer F by washing the bed at the same flow rate for 18 h. The stabilized bed height measured 32 cm. Protein solution (46.5 ml, 500 mg) from the 40 to 60% ammonium cut of S-200 (dialyzed against the column buffer) was loaded on the column at a flow rate of 32 ml/h and eluted with 700 ml of a 0 to 1 M KC1 gradient, in the same buffer system at the same flow rate. Five-milliliter fractions were collected and the absorbance of the fractions was monitored at 280 nM.
Phosphocellulose ChromatopraDhy-A ohosnhocellulose column (20.2 x-1.5 cm) was made followingthe Burgess procedure (14) and equilibrated with Buffer G. DEAE-Sephadex fractions showing RNase N activity were pooled, dialyzed against Buffer G, applied to the column, and eluted by a 0 to 0.5 M potassium phosphate gradient, pH 7.5, 250 ml, at a flow rate of 18 ml/h, collecting 4-ml fractions. Gel Filtration-A Sephadex G-150 gel bed (53 x 1.5 cm) was equilibrated with Buffer G at a flow rate of 8 ml/h. Phosnhocellulose column fractions showing RNase N activity were pooled and concentrated. The concentrated sample was dialyzed against Buffer G and applied (2.5 ml) to the gel. Elution was performed with the same buffer at the same flow rate (8 ml/h) and 2.5-ml fractions were collected.

N from E. coli
Storage of Enzyme-The enzyme from the last step of purification could be stored frozen at -20" without any detectable loss of activity for over 4 months. For detailed studies of the enzvme characteristics samples frozen in small aliquots were thawed ai approximately 0", dialyzed against assay buffer, and kept in ice for a period of 4 to 6 weeks.
Assay of RNase Activities-All assays essentially consisted of a Tris/HCl or potassium phosphate buffer (pH 7.5), enzyme from different sources, labeled substrates (in a volume of 5 ~1 in either Buffer C or D), and salt solution at the desired final concentration. The mixture (in 50 ~1 volume) was incubated at the desired temperature for an appropriate length of time with 20 pg of RNA in ribosomes, except if otherwise stated. For determining blank counts no enzyme control incubations were performed. Reaction was terminated by adding l/s volume of 0.5% SDS,' 12 rnM EDTA, 50% sucrose, and 0.1% bromphenol blue mixture. Sucrose helped to layer samples on polyacrylamide gels and bromphenol blue served the purpose of a marker in gel electrophoresis. For assays with ribosomes as substrate, samples were incubated at room temperature for 30 min. prior to application on the gel, to dissociate RNA from protein. Gel Electrophoresis-RNA samples were analyzed on a 5 to 12% tandem polyacrylamide gel (15) or a 5 to 15% tandem polyacrylamide gel. For most studies the bottom 4 cm of the gel contained 15% polyacrylamide, 0.4% methyl bisacrvlamide. and the ton laver contained 5% polyacrylamide, 0". 133% methyl bisacrylamide in aslab gel system (100 x 145 x 1.5 mm (15)). Gels were nrerun at 80 V for 0.5 h. then run with samples for 0.5 h at the same ioltage, and another 2.5 to 3 h at 150 V. Other conditions of electrophoresis were the same as described previously (3,15). Quantitation of RNA (large or smaller species) was done as detailed before (31. This gel system could retain large RNA at the origin, while pieces smaller than 5 S entered into the 15% gel. The interphase and the 5% gel contained various intermediate species. For quantitation, each region of the gel was cut and counted, values were corrected for background (no enzyme), and the totals normalized.
Also for some experiments samples were analyzed in tube gels (10 x 0.5 cm). After electrophoresis, gels were cut into 0.5~cm pieces, digested with H,O, and radioactivity was determined (31. The products of RNase N digest of 32P-labeled ribosomes were analyzed by a two-dimension polyacrylamide gel electrophoresis after Varricchio and Ernst (16). Samples were run in the first dimension, 15% gel with 7 M urea at pH 8.3, followed by an identical second dimension gel without urea. The bottom region of the first dimension gel, showing apparently a single distinct spot on the autoradiogram, was cut out and placed on the second dimension gel. Electrophoresis conditions were the same as for the first dimension. The gel was dried after electrophoresis and an autoradiogram prepared as usual. This gel system has been used successfully by others (16)  Five micrograms of each of the mononucleotides, 5'-UMP, 2':3'-cyclic UMP, 2'-UMP, and 3'-UMP, were applied individually or in a mixture. with and without an enzvme reaction mixture, which was incubated for 2 h at 40". In this system the RF of 5'-UMP is 0.227 and 2':3'-cyclic UMP is 0.700. Both 2'-UMP and 3'-UMP have the same R, of 0.500. The mobilities of the standard samples were not affected by the presence of the components from the enzymatic digestion.
['4C1uracil-labeled ribosomes were incubated with RNase N under conditions which gave approximately 60% acid-soluble products and applied on the chromatography paper (a no enzyme control was also included).
Both reaction and control were applied in the same way to the paper and the chromatogram was developed as described.
Spots containing nucleotides were identified under ultraviolet light after drying the chromatography paper at 45" for 30 min. Pieces of 1 cm each were cut and counted in toluenebased scintillation fluid for determining radioactivity.

Isolation of Enzymes
We could detect rRNA degradative activities in the S-200 as well as in the ribosome wash. In Fig. 1  Fractions showing RNase N activity on the phosphocellulose column were pooled, concentrated, dialyzed, and filtered through a Sephadex G-150 column. While most of the protein was eluted in a large peak, fractions eluted between 40 and 53 ml contained relatively little protein but showed very intensive RNase N activity. Those fractions were combined and used as the enzyme in all of the subsequent studies reported here. Determination of the specific activity of RNase N is rather complex with unpurified material because of the additive effect of other enzymes like RNase II and polynucleotide phosphorylase etc.
Purity of the protein material from the various fractions was determined by analysis in polyacrylamide gels according to the procedure of Davis (17). The last step in the purification decreased greatly the complexity of the proteins in the enzyme fraction. The enzyme preparation contained only three proteins, two major and one minor.

Requirements of Ions and Inhibition by Divalent Cations-
Since the substrate (ribosomes) is rather complex, it did not seem desirable to remove all of the ions from the assay mixtures. Thus, in all of the assays there was 0.1 mM magnesium acetate. As shown in Table I, monovalent cations enhance the reaction at 0.1 M. All monovalent cations tested seemed to be equally effective, Na+, K+, NH,+, and Tris. At higher salt concentration (0.2 M and above) the enzyme activity is reduced.
Mg2+ and Mn2+ inhibited the reaction almost completely at 20 mM. The presence of 2 mM Mn2+ or 8 mM Mg2+ in the assay mixture caused 80% inhibition of the reaction. pH Optimum-The pH optimum was tested by using a variety of buffers at a pH range of 4 to 10. The enzyme is active at a broad range of pH values (5 to 9) and its maximal activity is at pH 8 to 9.

Temperature
Optimum-The temperature optimum of the reaction was retested with the more purified preparation of RNase N (Fig. 3). Consistent with the less purified enzyme preparation (see Fig. 2) higher temperatures, 40-50", were more effective.
Heat Znactivation -The enzyme is rather heat-stable but it can be irreversibly inactivated by heating at relatively high temperatures. After being heated at 70" for 30 min the enzyme activity was completely lost, while after heating at 55" for 30 min it retained 25% of its activity.
Kinetics-The kinetics of the reaction was tested at 45". It was seen that the large molecules disappeared rather rapidly: in 15 min 60% of the substrate became smaller, while during the same time only 20% of the substrate entered the 15% gel, i.e. was degraded to small oligonucleotides.
Within 1 h most of the substrate was degraded to smaller pieces but only 60% of the substrate was in short oligonucleotides and nucleotides.
Substrate Specificity-The specificity of the enzyme was tested. An assay was carried out at different temperatures using rRNA as the substrate. At higher temperatures the activity increased. However, in these assays the increase of activity with temperature was found to be more monotonic and there was no discontinuity as observed when ribosomes were used as a substrate (see Fig. 3). This suggests that particularly at low temperatures (37" and lower) there is some protection of the rRNA by the ribosomal proteins-i.e. less accessibility of sensitive sites to RNase N.
Synthetic single stranded RNAs like poly(U) and poly(C) are also substrates for RNase N. At 40", 2.2 pmol of 23 S and 16 S rRNA were hydrolyzed/h by 1 pg of RNase N. Under the same conditions of assay 0.5 pmol of poly(U), M, = 1 x 105, or 0.66 pmol of poly(C), M, = 3.0 x 104, was hydrolyzed/h by 1 pg of RNase N. Hydrolysis of 23 S or 16 S RNA from the ribosomes or purified rRNA was almost at the same rate at 40". (Measurements were made for the diminution in the size of the substrates.) The above figures are first approximations.
It is rather difficult to carry out precise quantitation because of the complexity of the reaction and also the complex nature of the substrates themselves. Molecular weight of the synthetic RNA homopolymers was determined from their relative mobilities on the gel using rRNA as internal standards, but the homopolymers were not uniform in size and as soon as some cuts are being made it is possible that more substrate is created for the enzyme. 32P-labeled E. coli DNA when tested as a substrate for RNase N was not degraded to any detectable extent.

Mode of Action-
The level of small molecules formed at different temperatures (40, 45, and 50"; see Fig. 3) is proportional to the rate of disappearance of large molecules when substrate is not limiting, but the amount of small molecules accumulating is small as compared to the amount of large RNA disappearing. Also, the appearance of intermediate molecules is not proportional to the rate of disappearance of large molecules. These phenomena could be explained by assuming that RNase N is either an endonuclease or a nonprocessive exonuclease but not a processive exonuclease (18, 19).
To clearly distinguish a nonprocessive exonuclease from an endonuclease, the following experiment was carried out. The level of enzyme was left constant while the level of substrate was increased (keeping the same input counts in all assays by using a labeled substrate preparation and diluting it with an appropriate amount of unlabeled substrate) and the assay was carried out for a constant length of time (120 min). As can be seen in Fig. 4, when higher levels of nonlabeled substrate competed with a constant amount of labeled substrate, although most of the large molecules disappeared from the origin of the gel the level of material accumulating in the 15% part of the gel decreased (some of the material was nucleotides, see below). These results indicate that the enzyme is not an exonuclease. Should it be an exonuclease, the level of labeled nucleotides accumulated should have been proportional to the level of large molecules disappearing.
Since 3zPlabeled E. coli DNA was not a substrate for RNase N. these Ribonuclease N from E. coli 7673 experiments suggest that the enzyme is a nonspecific endoribonuclease . Products-In order to define the terminal products of the reaction, an exhaustive digestion of 3*P-labeled rRNA was run on a 5 to 15% polyacrylamide tandem gel with 7 M urea. The bottom of the 15% part of the gel was cut out and rerun on a similar gel system without urea (see "Methods"). Again all of the material migrated to a single spot (Fig. 51, indicating that the final products are rather homogenous and very likely to be small oligonucleotides or nucleotides. The product(s) which runs as a single spot at the bottom of the gel in the first dimension was found to be soluble in 5% trichloroacetic acid.
In order to characterize the product further, [i4C]uracillabeled ribosomes were used as substrate and after exhaustive digestion with RNase N the products were subjected to paper chromatography under conditions which clearly separate 2':3'cyclic nucleotides from 5'-and 3'-mononucleotides.
The results of one such experiment are summarized in Fig. 6. While even after prolonged incubation only a small percentage of the products migrated into the paper (most remained in the origin), almost all of the product which left the origin clearly comigrated with 5'-UMP and not at all with the 3'-mononucleotide or the 2':3'-cyclic nucleotide. Thus, it is evident that the enzyme preparation is capable of degrading rRNA, at least partially, to 5'-nucleoside monophosphate( In the experiment shown in Fig. 5 only about 8% of the total counts cochromatogrammed with 5'-UMP, while 70% of the counts were found to be acid soluble (5% trichloroacetic acid). This suggests that small oligonucleotides were among the reaction products. This again indicates that the enzyme is an endonuclease. The fact that the mononucleotides produced after exhaustive diges- FIG. 5. Analysis of product by two-dimensional gel electrophoresis. Lane 1, sample after digestion of 20 pg of 32P-labeled RNA in ribosomes with 4 pg of RNase N preparation, at 45" for 3 h, was run in a 5 to 15% urea gel (16). Lane 2, the bottom region of the first dimension gel was cut from the wet gel and rerun in the second dimension without urea. Lane 3, ribosome substrate incubated without enzyme on the second gel. For details see the text. The above figure is a composite presentation of two different autoradiograms from first and second dimension gels. The autoradiograms were developed after exposing gels to the films for different lengths of time.
tion with this enzyme are 5'-mononucleotides suggests that the oligonucleotides produced by this enzyme contain phosphates at their 5' end and hydroxyl groups at their 3' end. The inset in Fig. 6 shows an analysis of an identical sample (after exhaustive digestion with the enzyme) analyzed on a 5 to 15% tandem polyacrylamide gel, as described previously. Again, we can see that most of the digestion products migrate as oligonucleotides (see Fig. 5).

DISCUSSION
The studies presented in this paper show that Escherichia coli contains a nonspecific endoribonuclease, RNase N. Another known enzyme ofE. coli, RNase I (201, resembles RNase N to some extent, especially with respect to substrate nonspecificity and endonucleolytic activity. This enzyme is periplasmic (21) and its physiological function has not yet been established. The enzyme RNase N is distinguished from RNase I in at least two respects, it does require monovalent cations for its activity, while RNase I does not, and its final products are 5'-mononucleotides, while the products of RNase I are cyclic mononucleotides.
There are certain features which distinguish RNase N from other known E. coli ribonucleases. RNase III is an endoribonuclease specific for double stranded RNA. Moreover, it has been demonstrated that RNase III cannot attack rRNA (22) or single stranded RNA, and its final products are oligonucleotides (23,24). RNase II in strain N7060 (from where RNase N was isolated) is thermolabile and it has been previously demonstrated (10) that it could be completely inactivated by heating the enzyme solution at 50" for 10 min. RNase N is stable under these conditions, and the optimal temperature for its activity against rRNA is 45-50". Both RNase III and RNase II activities require Mg*+, while it is inhibitory for RNase N activity.
RNase N is probably an intracellular enzyme, but further studies are necessary to establish this point. The fact that it can be found, after cells are opened, in the ribosome pellet or 100 DISTANCE (cm) FIG. 6. Analysis of products by paper chromatography. ['*ClUracil RNA (20 pg) in ribosomes was incubated with 4 pg of RNase N preparation in 0.1 M Tris/HCl, pH 7.5, at 45" for 120 min and products analyzed by paper chromatography as described in the text. One-centimeter pieces, along each lane in the paper, were cut and counted in toluene-based scintillation fluid. Counts in the first 1 cm in the plot include also a region 1 cm below the base-line, where samples were applied on the paper. A time zero incubation sample was run on the same paper and counted. Its values (30 to 40 cpm) were used to assess the background counts. The plot was corrected for these background counts. The inset in the figure shows an identical sample (after digestion with RNase N) on a 5 to 15% tandem gel, as described previously. Mononucleotides migrate at the very bottom region, between 9.5 and 10 cm.

7674
Ribonuclease N from E. coli in the supernatant does not suggest a precise physiological location, since association with the ribosome pellet or with the supernatant is not necessarily its in situ location (25). However, this distribution between ribosome and supernatant could be meaningful and would place this enzyme in a similar physiological category as elongation factors involved in protein synthesis. Such a location would fit perfectly with its putative role in turnover of rRNA and a possible role in decay of mRNA.
Since it is a nonspecific endoribonuclease, it could be easily envisaged that RNase N would start to decay mRNA (see for instance Refs. 5,26,27,and below). Thus far no intracellular endoribonuclease has been detected in E. coli which could cleave nonspecifically single-stranded RNA in the cell (see Refs. 5,6,and 28). RNase N seems to fill this gap. Since it can attack a variety of RNAs one must envisage protection of the various RNAs from such an activity (26). Evidence is accumulating for a role for ribosomes as protectors for mRNA* (26), and, as discussed by Kaplan and Apirion (3), the rRNA is not accessible to ribonucleases in the cell in polysomes or in monosomes. The ribosome substrate used in these studies is most likely in the subunit form due to the extensive washes with high salt concentrations. Moreover, the enzyme is more active in the absence of divalent cations, an ideal condition to keep ribosomes in subunits. If RNase N participates in the process of turnover of ribosomes (obviously mutant analyses should be very useful in this respect) then the requirements for RNase II and polynucleotide phosphorylase in the process, as postulated from in uiuo studies (3, 291, could be well understood, since the production of nucleotides by RNase N alone is rather slow. Moreover, the few per cent of material co-migrating with 5'-UMP (Fig. 6) could be due to a contaminating exonuclease. (FlIrther analysis with a more purified preparation of RNase N would be necessary to settle this point.) An activity which degrades rRNA only to a limited extent was detected in ammonium sulfate fractions from the ribosomal wash (Fig. 1) and in protein fractions eluted from the phosphocellulose column (see "Results"). That this activity is different from RNase N is rather clear from the fact that their mode of action is dissimilar. When about the same level of large substrate disappears from the origin of the gel, the picture as revealed from the autoradiogram of the gel is very different for both activities (Fig. 1). Moreover, even after * E. Schneider, M. Blundell, and D. Kennell, personal communication.
exhaustive digestion it failed to convert most of the substrate (rRNA in ribosomes) to small oligonucleotides and nucleotides. In addition, the presence of 1 to 2 mM Mg2+ and Mn2+ inhibited the decay of the substrate. Thus, it is unlikely that this activity is RNase III which requires Mg2+ for its activity. Morevoer, RNase III was reported to be incapable of degrading rRNA (22). Further characterization of this activity is obviously in order.