Proc. Natl. Acad. Sci. USA Vol. 81, pp. 4290-4293, July 1984 Biochemistry Ribonuclease T: New exoribonuclease possibly involved in end-turnover of tRNA (Escherichia coli/3' terminus) MURRAY P. DEUTSCHER, CHRISTOPHER W. MARLOR, AND RICHARD ZANIEWSKI Department of Biochemistry, University of Connecticut Health Center, Farmington, CT 06032 Communicated by M. J. Osborn, March 26, 1984

ABSTRACT Examination of double mutants lacking one provide evidence that it is distinct from the previously de- of the exoribonucleases, RNase II, RNase D, RNase BN, or scribed exoribonucleases of E. coli. RNase R, and also devoid of tRNA nucleotidyltransferase has suggested that none of these RNases participates in the end- MATERIALS AND METHODS turnover of tRNA. This prompted a search for and identifica- Bacterial Strains. All strains used in this study are E. coli tion of a new exoribonuclease, termed RNase T. RNase T K-12 derivatives. The RNase II- strain, C3b; the tempera- could be detected in mutant Escherichia coli strains lacking as ture-sensitive RNase Dts strain, C3c; the RNase BN- strain, many as three of the known exoribonucleases, and it could be CAN; and its parent strain, CA265, have been described separated from each of the four previously described RNases. (10-12). C3 and CAN derivatives containing an rnd deletion, RNase T is optimally active at pH 8-9 and requires a divalent strains C3/5 and CAN/1, were prepared by using Pl-mediat- cation for activity. The enzyme is sensitive to ionic strengths > ed transduction to transfer the deletion from strain RB14 50 mM and is rapidly inactivated by heating at 45°C. Its pre- (13). For this purpose a TnJO transposon was inserted adja- ferred substrate is tRNA-C-C-[14C]A, with much less activity cent to the deletion, and tetracycline-resistant transductants shown against tRNA-C-C. RNase T is an exoribonuclease that were assayed for the absence of RNase D activity. The initiates attack at the 3' hydroxyl terminus of tRNA and re- RNase R- strain, S296-6808, was constructed by reintroduc- leases AMP in a random mode of hydrolysis. The possible in- ing rnb+ into strain S296-680 obtained from David Schles- volvement of RNase T in end-turnover of tRNA and in RNA singer (Washington University), using cotransduction with metabolism in general are discussed. trp+ (6), and assaying for transductants containing RNase II activity. In recent years it has become apparent that cells contain a Strain CAN/20-12 (lacking RNase II, RNase D, and multitude of distinct ribonucleases that participate in the var- RNase BN) and strain CA265/3-1 (lacking RNase II and ious degradative and processing reactions of RNA metabo- RNase D) were constructed by introduction of the rnb and lism (see ref. 1 for examples). Even in the case of exoribonu- rnd mutations into strains CAN and CA265. The strains were cleases, which generally have been considered to be nonspe- first made trp- by transduction of the trp: :TnJO marker from cific degradative enzymes, it is now clear that a variety of strain NK5151 (obtained from Nancy Kleckner, Harvard these activities with different specificities are present in a University). The rnb mutation was introduced by cotrans- single cell. Thus, four exoribonucleases have been identified duction with trp+ using P1 grown on strain C3b to generate previously in Escherichia coli (a fifth one that also may be strains CAN/20 and CA265/3. The rnd deletion was then considered is polynucleotide phosphorylase; ref. 2) on the transferred into these strains (as described above for con- basis of both genetic and biochemical criteria. These include struction of C3/5) to generate strains CAN/20-12 and the enzymes, RNase II (3), RNase D (4), RNase BN (5), and CA265/3-1. A-further derivative of strain CAN 20-12, also RNase R (5, 6). lacking RNase I, was constructed by first isolating a tetracy- Although the in vitro specificities of several of these en- cline-sensitive derivative of the strain (14) and then introduc- zymes have suggested possible functions (2, 3, 5, 7), in no ing rna by cotransduction with zbd-279::TnlO present in case has a physiological role been conclusively demonstrat- strain SK2255 (obtained from the Coli Genetic Stock Center, ed. One in vivo function that is thought to require exoribonu- Yale University). Extracts of this strain, CAN 20-12E, were clease action is the -C-C-A end-turnover of tRNA (8). In an devoid of RNase I, RNase II, RNase D, and RNase BN ac- attempt to determine which, if any, of the known exoribonu- tivity. cleases might be involved in this process, we have examined Derivatives of the various RNase- strains lacking tRNA the state of the 3' terminus in tRNAs isolated from strains nucleotidyltransferase (cca mutants) were constructed by deficient in each of these enzymes (9-11) and also lacking cotransduction of the cca locus from strain 35-10 with toiC tRNA nucleotidyltransferase so that defective -C-C-A se- as described (15). quences would not be repaired (8). To our surprise, in no Strains were grown in YT medium (16), usually supple- case did the absence of an RNase affect the amount of defec- mented with 0.4% glucose. Tetracycline-resistant strains tive tRNA in the double-mutant strains, suggesting that an- were grown in medium containing tetracycline at 10 ,ug per other nucleolytic activity might be involved in the end-turn- ml. over process. Substrates. E. coli tRNA was isolated by phenol extraction Accordingly, we investigated whether an additional exori- and isopropanol fractionation as described (17). tRNA-C-C- bonuclease might be present in extracts of E. coli that could [14C]A, tRNA-C-[14C]U, phosphodiesterase-treated [32p]- remove the terminal AMP residue from tRNA. In this paper tRNA, and [32P]rRNA were isolated or synthesized as re- we describe the identification of this nuclease, termed ported (4, 13, 18). [3H]Poly(A) and nonradioactive poly(A) RNase T, present an initial description of its properties, and were purchased from Miles. Other Materials. Radioactive nucleoside triphosphates The publication costs of this article were defrayed in part by page charge were obtained from Schwarz/Mann. Ultrogel AcA44 was payment. This article must therefore be hereby marked "advertisement" from LKB. Rabbit liver tRNA nucleotidyltransferase was in accordance with 18 U.S.C. §1734 solely to indicate this fact. purified as reported (19). All salts were reagent grade.

4290 Downloaded by guest on October 2, 2021 Biochemistry: Deutscher et aL Proc. Natl. Acad. Sci USA 81 (1984) 4291

Pieparation of Extracts and Gel Filtration. High-speed su- Table 2. Activity against tRNA-C-C-['4C]A in extracts prepared pernatant (S100) fractions for gel filtration were prepared from RNase- strains after rupture of the cells in an Aminco French press as de- tRNA-C-C-[14C]A hydrolysis, scribed (13). In some experiments cells were opened in buff- Strain Relevant phenotype nmol/15 min er B (50 mM glycine-NaOH, pH 9.0/0.1 mM dithiothrei- tol/0.1 mM EDTA). For small-scale experiments, cells were CA265 RNase' 0.47 ruptured by sonication. Gel filtration on Ultrogel AcA44 was CAN RNase BN- 0.44 carried out in 10 mM Tris chloride, pH 7.5/0.1 mM CAN/20 RNase BN-, II 0.41 EDTA/0.1 mM dithiothreitol/0.1 mM phenylmethylsulfonyl CAN/20-12 RNase BN-, II, D- 0.30 fluoride/1 M KCl/10% (vol/vol) glycerol. Cells were grown to A550 of 1, removed from thb growth medium Assays. The level of defective 3' termini in tRNA isolated by centrifugation, and resuspended in one-fourth the volume in 50 from various mutant cells was determined from the amount mM glycine-NaOH (pH 9.0). Cells were ruptured by sonication, and of [14C]AMP that could be incorporated in the presence of cell debris was removed by centrifugation. Aliquots of 100 Al were purified tRNA nucleotidyltransferase as described (20). assayed in 200-1.d reaction mixtures containing 50 mM glycine- Activity of the various RNases was measured under opti- NaOH (pH 9.0), 5 mM MgC12, and 53 ,ug of tRNA-C-C-['4C]A at mal conditions by determination ofacid-soluble radioactivity 370C for 15 min. Acid-soluble radioactivity was measured as de- released from different substrates (4). Standard assays were scribed. carried out in 100-,ul reaction mixtures: 20 mM glycine- zyme that might remove the 3' AMP residue from tRNA. NaOH, pH 8.9/5 mM MgCl2 containing 45 jig of diesterase- Identification of RNase T. To facilitate detection of a dis- treated [32P]tRNA for RNase D; 20 mM Tris chloride, pH tinct RNase that could act upon tRNA-C-C-[14CIA, we pre- 7.5/5 mM MgCl2/100 mM KCl/1 mM [3H]poly(A) for RNase pared extracts from a series of mutant cells, each lacking an II; 20 mM Tris chloride, pH 8.0/0.25 mM MgCl2/300 mM additional RNase compared to its parent (Table 2). The ab- KCl containing '70 ,ug of [32P]rRNA for RNase R; 20 mM sence of RNase BN led to about a 7% decrease in hydrolysis Hepes-NaOH, pH 6.5/0.2 mM CoCl2/200 mM KCI contain- of tRNA-C-C-['4CJA, and a similar decrease was found upon ing 37 ,ug of tRNA-C-['4CJU for RNase BN; and 50 mM gly- the further removal of RNase II. In a strain also lacking cine-NaOH, pH 8.9/5 mM MgCI2 containing 34 ,ug of tRNA- RNase D, an additional 20% of the activity was lost; howev- C-C-[14C]A for RNase T. er, this latter strain-CAN/20-12, which is devoid of RNase BN, RNase II and RNase D activity-still retained almost RESULTS two-thirds of the wild-type activity against tRNA-C-C- State of 3' Termini in tRNAs from RNase-/cca- Double [14CIA. These results implied the existence of another Mutants. We have shown (20) that about 10-15% of the RNase in the extracts that was the predominant activity tRNA population isolated from cca- cells contain incom- against this substrate under the assay conditions used. plete 3' termini that arise as a consequence of the end-turn- Identification of this activity was obtained by chromatog- over process known to occur in vivo (8). We reasoned that in raphy of an S100 fraction from strain CAN/20-12E (RNase cells that lacked the RNase responsible for removal ofthe 3'- BN-, II-, D-, I-) on Ultrogel AcA44 (Fig. 1). A single peak terminal residues, tRNA molecules would be largely- intact, of activity against tRNA-C-C-[14C]A was observed that elut- even in the absence of tRNA nucleotidyltransferase. Ac- ed at a position corresponding to a molecular weight of cordingly, double mutants were constructed that lacked a -50,000, assuming a globular protein. Also shown is the ac- particular exoribonuclease and tRNA nucleotidyltransfer- ase, and the state of the 3' termini in tRNAs isolated from these cells was evaluated by the amount of [14C]AMP that could be reincorporated into these molecules. Removal of all of the known exoribonucleases did not de- crease the level of defective tRNA present compared to a cell that contained all the nucleases (Table 1). In fact, even in a strain lacking three of these enzymes (CAN/20-12), the I amount of defective tRNA was unaffected. In all cases, the c 0 E presence of defective tRNA was dependent on the cca muta- CIO 0 tion. These findings suggested that the end-turnover of E tRNA did not involve RNase II, RNase D, RNase BN, or E C RNase R and prompted us to search for an additional en- .0 ._.0 Table 1. State of 3' termini in tRNA isolated from cca+/RNase- 0 20 and cca-/RNase-mutant strains AMP incorporation, z nmol/mg of tRNA ccE cc Strain Relevant phenotype cca+ strain cca- strain ES A19 RNase+ 0.1 3.2 z C3b RNase II- <0.1 3.1 C3c RNase DtS <0.1 3.6 C3/5 RNase D- <0.1 3.5 <0.1 3.3 CAN RNase BN- Volume (ml) S296-6808 RNase R- 0.1 4.1 RNase II, BN- 0.2 4.3 CAN/20-12 D-, FIG. 1. Gel filtration of an S100 fraction from strain CAN/20- tRNA was isolated from each of the indicated strains, and 12E. A concentrated, high-speed supernatant fraction was prepared, [14C]AMP incorporation into 100 jg was determined with purified and 5.4 ml was chromatographed on a column of Ultrogel AcA44 (92 tRNA nucleotidyltransferase as described. A19 data are taken from x 2.5 cm) as described. Fractions of 4.63 ml were collected. Ali- an earlier publication (20). tRNA was isolated from strain C3c after quots of 5 ,ul and 20 ,u1 were assayed against tRNA-C-C-[ 4C]A and the cells were kept at 45°C for 1 hr. ts, Temperature sensitive. [32P]rRNA, respectively. Downloaded by guest on October 2, 2021 4292 Biochemistry: Deutscher et al. Proc. NatL Acad Sci. USA 81 (1984)

tivity against [32P]rRNA, which defines the other known ex- activity and one of the other exoribonucleases were chtoma- oribonuclease still remaining in this strain, RNase R. The tographed on Ultrogel AcA44, and the elution positions of presence of two peaks of activity able to hydrolyze [32p; RNase Tand the wild-type form of each of the enzymes were rRNA has been observed repeatedly in the CA265 gehetic compared (Fig. 2). From analysis of strain C3b (RNase I, background; the identity of the lower molecular weight peak II-) (Fig. 2A), it can be seen that RNase T separated and was is unknown, although its properties suggest that it is related slightly larger than RNase D. Likewise, RNase T separated to RNase R. Nevertheless, these results show that the en- from RNase II upon chromatography' of an S100 fraction zyme hydrolyzing tRNA-C-C-['4C]A is distinct from either from CAN/1 (RNase BN-, D-) (Fig. 2B), and it separated of those two activities. In. addition, its elution position dif- from RNase BN in a S100 fraction from strain CA265/3-1 fered from each of the other known exoribonucleases (see (RNase D-, II-) (Fig. 2C). These data confirm that RNase T below). This enzyme was termed RNase T because its prop- is not a modified form of one of the previously described erties suggest that it may participate in turnover of tRNA. exoribonucleases but is a distinct enzyme. Inasmuch as RNase T was identified in a multiple-RNase- Properties'of RNase T. RNase T was purified from a strain mutant strain, it was necessary to ensure that this putative C3 derivative lacking RNase I, RNase II, and RNase D. The new enzyme was not a mutant form of one of the already properties described here were determined with an enzyme known RNases with an altered specificity. To explore this fraction that was purified -1,000-fold. Details of the purifi- possibility, S100 fractions from strains containing RNase T cation procedure will be described elsewhere. With tRNA-C-C-[14C]A as substrate, RNase T7 had a pH optimum in the range of pH 8-9, with higher activity in gly- cine-NaOH buffers than in Tris chloride. The enzyme re- quired a divalent cation for activity; the requirement could be satisfied by either Mg2', Mn2+, or Co2+. Highest activity

0) was obtained with Mg2+ in the range of 3-10 mM. RNase T - was strongly inhibited by salts at ionic strengths in excess of 2. 50 mM. It also was sensitive to heat inactivation, losing half a-- of its activity in 7 min at 450C. This latter observation may explain why RNase T was not previously detected; in earlier studies in which extracts from temperature-sensitive RNase EkE D strains were used, a heating step at 450C was included to inactivate RNase D. Currently, the use of RNase D deletion strains has eliminated the need for heating extracts. RNase T is an exoribonuclease. As indicated by paper chromatography, all the radioactivity released from tRNA- 'a C-C-[14C]A was in the form of the'mononucleotide AMP, which could be converted to adenosine upon treatment with E bacterial alkaline phosphatase (data not shown). In addition, RNase T digested tRNA-C-C-A in a randoih fashion and ini- 0 Z0 tiated attack at the 3' terminus of the substrate because, in E the presence of [14C]ATP and tRNA nucleotidyltransferase, a ).- . the enzyme promoted the exchange of radioactive AMP with 0 E the original unlabeled terminus (Fig. 3). This could occur - only if RNase T removed the terminal AMP residue from tRNA and then dissociated from the RNA chain to allow IxE tRNA nucleotidyltransferase to repair' the terminus with [14C]ATP. A similar exchange with [ 4C]CTP proceeded only 9 about 10% as rapidly (Fig. 2), suggesting a much slower rate of CMP removal from tRNA. This was confirmed directly by z measuring release of radioactivity from tRNA-C-[14C]C (see I_9 below); tRNA-C-Cp was inactive as a substrate in this reac- tion (Fig. 3), indicating that RNase T requires a free 3' hy- -0 droxyl group in order to initiate attack on an RNA chain. 3C-- A preliminary survey of the substrate specificity of RNase S.m T indicated that intact tRNA-C-C-A was the preferred sub- strate (Table 3). Removal of nucleotides (as in phosphodies- E terase-treated tRNA) or addition of the nucleotides (as in tRNA-C-C-A-C-C) reduced activity. Among substrates con- taining substitutions within the -C-C-A sequence, tRNA-C-A was more active than tRNA-C-U or tRNA-C-C. Poly(A) was 13 zvwV jV J 3V qvv inactive as a substrate. These data indicate that the substrate Volume (ml) specificity of RNase T differs from that of the other known exoribonucleases.

Gel from containing FIG. 2. S100 RNase T activity and other RNases. Extracts were prepared and DISCUSSION columns run as in Fig. 1 except that buffer B was used to prepare the The studies presented here provide evidence for the exis- extracts from strains C3b and CAN/1. (A) Strain C3b. Aliquots of 5 tence of an additional exoribonuclease, termed "RNase T," 1Ld and 10 /Ld were assayed for RNase T and RNase D, respectively. in extracts of E. coli. This brings to five (six, if one includes (B) Strain CAN,/1. Aliquots of 5 ul and 2 were assayed for RNase polynucleotide phosphorylase) the number of exoribonu- T and RNase II, respectively. The lowest molecular weight peak is cleases that have been identified in this cell. In view of this RNase I. (C) Strain CA265/3-1. Aliquots of 5 g1d and 20 1.l were assayed for RNase T and RNase BN, respectively. The low molecu- increasingly large number of similar activities, we have at- lar weight peak active on tRNA-C-['4C]U is RNase I. tempted to ensure that RNase T is a distinct enzyme. Thus, Downloaded by guest on October 2, 2021 Biochemistry: Deutscher et aL Proc. NatL Acad. Sci. USA 81 (1984) 4293

ends of the chains (8, 20). This fits very well with the activity displayed by RNase T in vitro. Because the studies with RNase/cca double mutants suggested that the other identi- fied exoribonucleases are not involved in end-turnover, RNase T must be considered the best possibility for carrying out this process in vivo. However, it should be pointed out E that it is still C unclear whether end-turnover of tRNA repre- 04 sents an important physiological process or whether it sim- ply is a side reaction that can occur when uncharged tRNA 0 comes in contact with the appropriate RNase (8, 20). As long 00 3 as a cell contains tRNA nucleotidyltransferase to rapidly re- pair the damage of tRNA, the cell would be unaffected. C e This, of course, raises the question of what the true phys- iological role of RNase T might be. Previous studies with 802 - mutants lacking other exoribonucleases have failed to find an in vivo function for any of these enzymes (5, 6, 13). Even z the triple RNase mutant, CAN/20-12, described here, grows OjI normally. If, as suggested earlier (13), another exoribonu- clease can take over the function of one that is missing in a /[14CJCTP mutant strain, the existence of RNase T adds another en- 0~~~~~~~~ zyme to the complement of possible substitutes. The pres- ence of RNase TO 20 30 T could explain the continued viability of a Time (min) strain lacking RNase II, RNase D, and RNase BN, especial- ly since RNase T does have some overlapping specificity FIG. 3. Exchange of radioactive nucleotides into tRNAs in the with these other enzymes. presence of RNase T and tRNA nucleotidyltransferase. Reaction mixtures were set up as usual for RNase T assays except that 100 ug We thank Ellen Petkaitis for construction of strain CAN/20-12E. of unlabeled tRNA-C-C-A or tRNA-C-Cp, 0.5 mM [14C]ATP or This work was supported by Grant GM16317 from the National In- [14C]CTP, and 0.2 unit of tRNA nucleotidyltransferase were also stitutes of Health and is no. 41 in the series "Reactions at the 3' present. After incubation at 370C for the indicated times, acid-pre- terminus of tRNA." Paper no. 40 is ref. 22. cipitable radioactivity was determined. The curve for tRNA-C-Cp was the same as with no RNase T. 1. Boyer, P. D., ed. (1982) The Enzymes (Academic, New York), Vol. 15, Part B. we have shown that RNase T is present in a variety of mu- 2. Littauer, U. Z. & Soreq, H. (1982) in The Enzymes, ed. Boyer, tant strains lacking as many as three of the previously identi- P. D. (Academic, New York), Vol. 15, Part B, pp. 518-553. fied RNases. We also have demonstrated that RNase T can 3. Shen, V. & Schlessinger, D. (1982) in The Enzymes, ed. Boyer, be from the forms of RNase RNase P. D. (Academic, New York), Vol. 15, Part B, pp. 501-515. separated wild-type II, 4. Ghosh, R. K. & Deutscher, M. P. (1978) J. Biol. Chem. 253, D, RNase BN, and RNase R on the basis of size. In addition, 997-1000. the chromatographic properties of RNase T during its purifi- 5. Asha, P. K., Blouin, R. T., Zaniewski, R. & Deutscher, M. P. cation differed from those of the other known enzymes (un- (1983) Proc. Natl. Acad. Sci. USA 80, 3301-3304. published observation). The size and catalytic properties of 6. Kasai, T. K., Gupta, R. S. & Schlessinger, D. (1977) J. Biol. RNase T most closely resemble those of RNase D (21). Chem. 252, 8950-8956. However, whereas RNase D is relatively inactive against in- 7. Cudny, H. & Deutscher, M. P. (1980) Proc. Natl. Acad. Sci. tact tRNA (4, 21), it is the preferred substrate of RNase T. USA 77, 837-841. Furthermore, RNase T is present in strains containing an 8. Deutscher, M. P. (1983) in Enzymes ofNucleic Acid Synthesis RNase D deletion mutation and it is much more sensi- and Modification, ed. Jacob, S. T. (CRC, Boca Raton, FL) (13), Vol. 2, pp. 160-183. tive to sulfhydryl inhibitors than is RNase D (unpublished 9. Nikolaev, N., Folsom, V. & Schlessinger, D. (1976) Biochem. observation). All of these points lead us to the conclusion Biophys. Res. Commun. 70, 920-924. that RNase T is a separate enzyme and not a manifestation of 10. Maisurian, A. N. & Buyanovskaya, E. A. (1973) Mol. Gen. a previously described activity. Genet. 120, 227-229. The specificity of RNase T suggests that it is a likely can- 11. Zaniewski, R. & Deutscher, M. P. (1982) Mol. Gen. Genet. didate for the RNase involved in end-turnover of tRNA. In 185, 142-147. vivo, turnover predominantly affects the terminal AMP resi- 12. Asha, P. K. & Deutscher, M. P. (1982) J. Bacteriol. 156, 419- due, with much less removal of the CMP residues, even in 420. 13. Blouin, R. T., Zaniewski, R. & Deutscher, M. P. (1983) J. the absence of tRNA nucleotidyltransferase to repair the Biol. Chem. 258, 1423-1426. Table 3. Substrate of RNase T 14. Maloy, S. R. & Nunn, W. D. (1981) J. Bacteriol. 145, 1110- specificity 1112. Nucleotide released, 15. Foulds, J., Hilderman, R. H. & Deutscher, M. P. (1974) J. Substrate pmol/5 min Bacteriol. 118, 628-623. 16. Miller, J. H. (1972) Experiments in Molecular Genetics (Cold tRNA-C-C-['4C]A 231 Spring Harbor Laboratory, Cold Spring Harbor, NY), p. 433. tRNA-C-[14C]A 36 17. Deutscher, M. P. & Hilderman, R. H. (1974) J. Bacteriol. 118, tRNA-C-['4C]C <5 621-627. tRNA-C-[14C]U <5 18. Deutscher, M. P. & Ghosh, R. K. (1978) Nucleic Acids Res. 5, tRNA-C-C-A-[14C]C-C 37 3821-3829. [32P]tRNA 347 19. Deutscher, M. P. (1972) J. Biol. Chem. 247, 450-458. Diesterase-treated [32P]tRNA 85 20. Deutscher, M. P., Lin, J. J.-C. & Evans, J. A. (1977) J. Mol. [3H]Poly(A) <5 Biol. 117, 1081-1094. 21. Cudny, H., Zaniewski, R. & Deutscher, M. P. (1981) J. Biol. Assays were carried out as described using RNase T assay condi- Chem. 256, 5633-5637. tions. Substrates were present at -0.3 mg/ml except poly(A), which 22. Solari, A. & Deutscher, M. P. (1983) Mol. Cell. Biol. 3, 1711- was at 0.4 mg/ml. 1717. Downloaded by guest on October 2, 2021