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 September 29, 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.