Volume 5 Number 11 November 1978
Nucleic Acids Research
An Escherichia coli ribonuclease which removes an extra nucleotide from a biosynthetic intennediate of bacteriophage T4 proline transfer RNA
Francis J.Sclunidt* and William H.McClain Department of Bacteriology, University of Wisconsin, Madison, WI 53706, USA Received 7 August 1978 ABSTRACT The biosynthesis of bacteriophage T4 tRNA Pr, tRNA Ser, and tRNAIle requires enzymatic removal of extra nucleotides from the 3' terminus of the respective precursor RNAs. A ribonuclease activity capable of catalyzing such reactions has been partially purified from uninfected Escherichia coli using an artificial precursor RNA as substrate. A number of ribonuclease activities were resolved during purification. Use of E. coli strain BN, a mutant known to be deficient in the relevant ribonuclease activity, permitted us to identify it in wild-type cells. This activity was designated the BN ribonuclease. BN ribonuclease had an apparent molecular weight of 35,000 as measured by Sephadex gel filtration. Mg2+ was required for activity, which was optimal at [Mg2+] of 2mM. Activity did not require monovalent cations K+ or Na+. BN ribonuclease was less efficient at removing extra residues in the biosynthesis of tRNASer and tRNAIle than in the biosynthesis of tRNAPro.
INTRODUCTION
Bacteriophage T4 codes for eight transfer RNA species. These tRNAs have furnished a useful system for the study of tRNA biosynthetic pathways and of the enzymes involved in these pathways. Three host enzymes required for T4 tRNA biosynthesis have been identified (1-6). Ribonuclease P cleavages generate the 5' ends of all mature T4 tRNAs. Transfer RNA nucleotidyltransferase (E.C. 2.7.7.25)synthesizes 3' terminal C-C-A sequences of T4 tRNA P tRNA , tRNA , and tRNA . Both enzymes have been partially purified from uninfected E. coli and shown to catalyze the appropriate reactions in vitro. A third host enzyme is required for biosynthesis of T4 tRNA , tRNA Ile . Extra nucleotides are removed from the 3' end of these tRNA and tRNA sequences before the C-C-A terminus is synthesized by tRNA nucleotidyltransferase. Removal of these residues does not occur in E. coli strain BN, and this deficiency has been attributed to loss of a ribonuclease activity (6, 7). In this communication, we report the partial purification and initial characterization of this ribonuclease, termed the BN ribonuclease. G Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
4129
Nucleic Acids Research MATERIALS AND METHODS Strains. Bacteriophage strains T4D and T2L as well as Escherichia coli strains B and BN were from our collection. Materials. Chromatographic resins were obtained from Whatman Products (DE52 DEAE cellulose) and Pharmacia, Inc. (Sephadex G25 and G100). [5- H]UTP (s.a. 20 ci/mmole) was obtained as the tetrasodium salt from New England Nuclear Corporation. Venom phosphodiesterase was from P-L Biochemicals. All other reagents were commercial analytical grade reagents. RNA sequence analysis. This was performed as described previously (1). Construction of tRNA-CU*. This analogue of immature tRNA was prepared by the procedure as outlined in Fig. 1 (8). Unfractionated E. coli tRNA (235 A260 units) in 0.55 ml of 0.04 M glycine-NaOH,pH 8.7,0.01 M Mg-acetate was equilibrated at 200, then venom exonuclease (0.1 mg in 0.1 ml of the above buffer) was added and the reaction allowed to proceed at 20° for 3 hrs. Digestion was terminated by phenol extraction of the reaction mixture and the product ("tRNA-C") was precipitated at -20° by addition of 25p4 SM K-acetate, pH5.0, and 2 ml absolute ethanol. Yield: 170 A260 units tRNA-C. A partially purified preparation of tRNA nucleotidyltransferase was used to add a single *5'UMP residue to tRNA-C. The reaction mixture contained in a total volume of 4.0 ml: 200 pmole Tris Cl, pH8.5, 80 pmole Mg-acetate, 60 pmole and S-mercaptoethanol, 100 pCi (Snmole) [5units tRNA nucleotidyltransferase. (One unit catalyzes the addition of 1 pmol/min of ATP to tRNA-C.) Reaction was at 370 for 3 hrs at which time the product tRNA containing [3H]UMP ("tRNA-CU*") was isolated by phenol extraction and chromatography on DE23 cellulose (4). Assay of BN RNase. The reaction mixture contained 1-3x10 cpm of tRNACU* and enzyme in 0.1 ml Buffer A (0.OlM Tris base/0.1 M NH 4Cl/0.02 M MgCl2/ 0.001 M dithiotheitol; this solution is self-buffering at pH 8.0). At the end of the incubation period, tRNA was precipitated by addition of serum albumin and TCA (8); then the precipitate was collected on Whatman GFA glass fiber filters, washed and counted. Alternatively an ethanol-salt mixture was used to precipitate the RNA (9) and the supernatant liquid was counted. Reactions of RNase with [ 32P immature tRNAPro were done as described previ-
3HUTP,
3x104
ously (6). Partial purification of BN RNase.
Frozen E. coli B
cells, 9.0 g,
were
partially thawed in a prechilled mortar on ice. The cell paste was mixed with 18.0 g levigated alumina and ground until a smooth paste was formed. Then Buffer A (9.0 ml) was mixed with the slurry and 100 vg DNase (Worthing4130
Nucleic Acids Research ton DPFF, RNase-free) was added. The mortar was kept on ice for 40 min. The slurry was centrifuged at 8000 xg for 19 min and the supernatant removed and centrifuged again at 30,000xg for 40 min. To this S30 supernatant (6.0 ml) was added 3.0 ml of 5.0% (w/v) protamine sulfate. The slurry was stirred on ice for 15 min and then centrifuged at 30,000xg for 30 min. To the resulting supernatant (8.2 ml) was added 2.45 g (NH4)2S04 (Mann, enzyme grade). The precipitate from this step was isolated by centrifugation and redissolved by addition of 1.5 ml Buffer A. This solution was divided into two aliquots which were desalted on a column of Sephadex G25 (0.9 cm x 29 cm in Buffer A). The combined desalted enzyme fractions were applied to a column (0.9 cm x 14 cm) of Whatman DE52 cellulose' in Buffer A. The column was washed with two column volumes of Buffer A and then with a linear gradient of 75 ml each of Buffer A and Buffer B (Buffer B is the same as Buffer A except that it also contains 0.2 M KC1). The peak enzyme fraction which eluted from DEAE cellulose beginning at 14 mmho or approximately 0.1 M KC1 (the second peak in Fig. 3, top) was further fractionated on Sephadex G200. Two ml of the DE52 fraction were applied to and eluted from a column of Sephadex G200 (0.9 cm x 60 cm) in Buffer A. Fractions of 1.0 ml were taken. RESULTS
Assay of BN Ribonuclease. In our previous work (6) this RNase activity was detected in crude extracts of E. coli by its ability to alter the 3' terminus of immature T4 tRNA ro, and the reaction was followed by fingerprint assay. Since fingerprint assays are expensive and time consuming,
they are unsuitable for use in enzyme purification. We therefore developed a rapid and simple assay using an artificial substrate (Fig. 1). This method utilized several reactions of tRNA, characterized by Carre and Chapeville (8), to produce an analogue of immature tRNA . First, treatment of bulk E. coli tRNA with venom exonuclease at low temperature removed the terminal C-A sequences from the tRNA chains. The resulting tRNA-C was a substrate for tRNA nucleotidyltransferase which in the absence of ATP and CTP added a single [5-3H]UMP (8). The product of the reaction (tRNA-CU*) was a modified tRNA ending C-U* instead of C-C-A, where the U* denotes a terminal [3H] uridine nucleoside. Because of its resemblance to immature (which ends C-U), we reasoned that tRNA-CU* might tRNA be a substrate for the processing RNase deficient in E. coli strain BN. The results in Fig. 2 show that an extract of wild-type E. coli B was 4131
Nucleic Acids Research venom tRN A-CCA OH tRNA-CCAOH
exonucleose tRNA-COH ~20 tRNA
nucleotidyl -fH] UT P
UMP+ *tRNACOH Fig. 1.
~~~BNRNose -
t RNA- CUOH
Scheme for assay of the BNl RNase.
100
Ea. 80
'Z soIa.
20 60-
E. coil BN
C e
40-
0
E. coil B
20
20
40
60
Time, min Fig. 2. Release of [3H] from tRNA-CU* by cell-free extracts. concentration in reaction mixtures was 10 mg/ml.
Protein
about twice as efficient in removing [5- H]U from the artificial substrate as This result meant that the artificial substrate could be used to assay the relevant ribonuclease. However, tRNA-CU* was a substrate for other nucleases since vig. 2 shows that some of the [3H]U was rendered acid soluble by the mutant extract. Partial purification of BN RNase. Throughout the purification of this enzyme, initial fractionation steps were assayed using tRNA-CU* as the substrate. Active fractions were then examined for ability to remove pU nucleotide from [ P]-labeled immature tRNA . The latter step was necessary because several cellular nucleases removed [ H]U from tRNA-CU*.
was an extract of E. coli BN.
4132
Nucleic Acids Research BN RNase activity did not associate with ribosomes when extracts were The activity could be purified prepared in the presence of 0.1 M NH4Cl. from either an IQ00 supernatant fraction or from the supernatant remaining after protamine sulfate precipitation of ribosomes and large nucleic acids. Ammonium sulfate fractionation experiments indicated that all BN RNase activ-
ity was precipitated by addition of 0.3 g/ml of (NH4)2S04 to either the S100 fraction or to the supernatant rema-ining after protamine sulfate precipitation.
The (NH4)2S04 precipitate was resuspended in Buffer A and desalted on Sephadex G25. The fraction was then applied to a column of Whatman DE52 DEAE cellulose. Elution was with a linear KC1 gradient (0-0.2 M) in Buffer A. Fig. 3 (top) shows that the majority of the wild-type (E. coli B) RNase activity was eluted from the column in two incompletely separated peaks. The RNase activity from E. coli BN, in contrast, eluted in only one peak (Fig. 3,
bottom), at the position of the first main peak in wild-type E. coli B. The second peak of wild-type RNase activity therefore probably corresponds to the RNase which is deficient in E. coli BN. In control experiments (data not shown) this second peak of wild-type activity removed a pU nucleotide unit
from
, while the residual RNase activity from P-labeled immature tRNA E. coli BN eluting at the same conductivity (beginning at 14 mmho) was incapable of removing this pU nucleotide. BN RNase was further separated from contaminating RNase activity by chromatography on Sephadex G200, as shown in Fig. 4. This procedure also
indicated that the molecular weight of BN RNase is approximately 35,000. BN RNase at this state of purification removed a single pU residue from Pro . Data allowing this conclusion are shown in Fig. 5, where immature tRNA the RNase A (pyrimidine-specific) fingerprints of immature tRNAPrO before and after BN RNase treatment are presented. The 3' terminal sequence of immature is G-G-A-G-A-C-U. RNase A digestion of tRNA terminating in this tRNA sequence gives G-G-A-G-A-Cp as the 3' terminal product (see Fig. 5a). (The other product from the 3' terminus of the tRNA, uridine, does not contain frac132pI phosphate and so is not observed by autoradiography.) The G200 Pro leavtion of BN RNase removed the terminal pU residue from immature tRNA ing a new 3' terminus, G-G-A-G-A-C; the position of this oligonucleotide in the RNase A fingerprint is shown in Fig. Sb. Fig. 6 shows how the sequence of G-G-A-G-A-C was characterized. Comparison of the amount of 3' terminus removed from immature tRNAPr by crude cell-free extracts (see Seidman, et al (6)) with that removed by BN RNase purified through G200 chromatography
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Nucleic Acids Research
A2-
0
cpm on
1200 E. coli BN
0.6
30-
600
A280 800
I 8004
20-
Conductivity, 10
cpm on filter AAA
mmho
A~~~~~ 0.2
10
10A 20
30
40
1200
FRACTION NUMBER Fig. 3. tions.
Chromatographic profiles on DE52 cellulose of the
(NH4)2S04
frac-
indicated an overall enrichment of between 50- and 100-fold. and The G200 fraction of BN RNase was incubated with immature tRNA the reaction mixture was spotted onto DEAE paper for electrophoresis at pH 3.5. Small products liberated in this reaction included all four 5' mononucleotides and short oligonucleotides 2-3 residues in length. The same products were found in greater yield when the peak of RNase activity eluting before BN RNase was examined. This activity may be RNase D, an enzyme of 60,000 molecular weight that apparently generates 5' mononucleotides from 4134
Nucleic Acids Research
0.2 0
A280
CD
0.1
41
-J
0
FRACTION NUMBER Fig. 4. Sephadex G200 chromatography of the DE52 fraction. The bir graph represents the molar yield of G-G-A-G-A-C following treatment of [ 2p] immature tRNAPro with 0.2 ml column fraction containing BN RNase (see Fig. 5).
tRNA chains (12).
It is possible that RNase D or a similar activity contami-
nates the BN RNase fraction as a result of incomplete column resolution. Al-
ternatively, the BN RNase may have a broad specificity. Further purification is needed to resolve this uncertainty. Properties of purified BN RNase. BN RNase purified through the Sephadex G200 step was exchanged with Buffer A lacking Mg by Sephadex G25 chromaof was addition reconstituted tography. Activity by differing concentrations of MgCl2 to the enzyme. The BN RNase fractions containing various concenion were assayed by reacting equal amounts of enzyme with trations of Mg 132pI immature tRNAPro and fingerprinting the RNA products. These experifor its activity (Fig. 7); maximal ments revealed that BN RNase requires Mg 2+ of 2mM. At concentrations above this level, activity was obtained at a [Mg 4135
Nucleic Acids Research a
I.h 46
40.-.
_Al
*%-CG,4,(--4Cp 1%.
G(,A 7AC0
.-.
2nd
I
0
'*'V
GG
AC.
*S
9
a
b
Fig. 5. RNase A fingerprint of immature treatment with purified BN RNase.
tRNAro
(a) before and (b) after
G-G -A-G-A-C
RNase T2 RN*eT Gp(3), Ap(2) Venom
pG(2), pA(2), pC
RNasw RNo- TiT1
Gp(2), RNose T2
A-Gp,
A-C
RNose T2 Gp
RNase T2 Ap, Gp
-
Venom
Fig. 6.
Ap
pC
Characterization of the new 5' terminal sequence, G-G-A-G-A-C.
markedly inhibited the enzyme so that the activity was reduced two2 fold at [Mg ] of 10 mM, and was barely detectable at 20 mM Mg Monovalent cations were apparently not required for activity. The activity of BN RNase as measured in fingerprint assays was not affected by exchange on Sephadex G25 with 0.01 M TrisCl, pH 8.0/0.002 M MgCl2 buffer concontaining either KC1, NaCl or no monovalent cation. However, the enzyme may
Mg
4136
Nucleic Acids Research
a
w
z 0.2
0
10-4
i0-3
2
[Mg2] M Fig. 7. Effect of [Mg 2+] on BN RNase activity as measured by the yield of G-G-A-G-A-C. Reaction mixtures contained 25 ,ug protein. have carried along a required cation from Buffer A to allow the reaction to proceed. Exhaustive dialysis of purified BN RNase against buffer A was not possible since activity was lost. Purif ied BN RNase activity was somewhat unstable at 0° C ., with a half-
life of 5-7 days. The enzyme could, however, be stored-frozen at -70° for at least one month without loss of activity. Reduced reaction with other potential substrates. Besides tRNA Potwo other bacteriophage T4 tRNA species require the removal of extra nucleotides prior to C-C-A synthesis . We isolated the appropriate [ 32p'L] labeled precursors and subj ected them to reaction under conditions which proved optimal .Reaction with these substrates was for removal of pU from immature tRNA less ef ficient . The tRNAPo-tRNA Srprecursor RNA requires removal of U-A-A from the 31' end of the molecule. The 3 ' terminal sequence of precursor RNA is C-C-U-C-CG-U-A-A; removal of pU-A-A in vivo generates a precursor RNA which ends C-c-C U-C-C-G (1). This reaction does not occur in E. coli strain BN (6). The reaction of BN RNase (purif ied through the DE52 column step) with precursor RNA was less complete than that with immature tRNA tRNA -
4137
Nucleic Acids Research tRNA
, since the molar yield of C-C-U-C-C-G was estimated to be 0.01-0.03;
resulted in a yield of G-G-A-G-A-C parallel reaction with immature tRNA equal to 0.5. Characterization of C-C-U-C-C-G included its position on the two-dimensional fingerprint (6) and the products of RNase T2 digestion. Similar results were obtained when BN RNase was reacted with T4 tRNA Ile Ile . The latter RNA species is pretRNA precursor RNA or immature tRNA P cleavage of the precursor RNA, though it has not sumably derived by RNase been determined if this cleavage precedes conversion of 3' U-A-U residues to C-C-A. Both of these RNAs terminate with U-C-A-U-A-U. In vivo, the BN RNase is required for removal of pU-A-U from the 3' end of the tRNA e sequence (6). When either RNA species was treated with BN RNase purified through the DE52 column step, removal of pU-A-U residues occurred to a limited extent (0.020.03 molar yield of U-C-A). The product U-C-A was identified solely by its position on the two-dimensional RNase T1 fingerprint.
DISCUSSION In the present work we have partially purified an enzyme, the BN RNase, Pro . This which removes a pU residue from the 3' terminus of immature tRNA reaction occurs in vivo in wild-type cells but not in mutant strain BN (6). As expected, BN RNase was not detected in strain BN. While purified BN RNase acted quite efficiently on immature tRNA
ro, it
was less efficient with immature tRNA Ile, the tRNA Pr-tRNASer precursor RNA
and the tRNA hr-tRNA precursor RNA. Removal of 3' nucleotides from the latter group of RNAs is also abolished by the BN mutation in E. coli (6). If the BN phenotype is the result of a single mutation, purified BN RNase should catalyze removal of 3' residues from all these RNA species. Several explanations for reduced efficiency can be mentioned. This reduced efficiency may result from a greater complexity in removing three nucleotides as opposed to Pro . If the probability of removing pU one nucleotide from immature tRNA is 0.5, then the probability of removing three resifrom immature tRNA 3 dues might be (0.5) =0.125. Alternatively, the BN RNase was purified from
uninfected E. coli; bacteriophage induced modification of the enzyme may occur. Finally, it is possible that the in vivo state of BN RNase is important for its action. Assay methods for processing ribonucleases of tRNA precursors have previously relied on two types of measurements, either structural changes (e.g. cleavage) of isolated precursor RNAs (10) or the production of a functional tRNA product which can itself be measured (11). This paper presents 4138
Nucleic Acids Research third approach which, because of its relative simplicity,
a
for
a
variety of
enzymes
may prove
useful
of this type.
ACKNOWLEDGEMENTS We thank Prof. Robert M. Bock for suggesting the
substrate in assaying the work
was
enzyme
use
of
an
artificial
and for other helpful discussions.
This
supported by NIH grant AI10257 and NIH Research Career Development
Award AI10002 to W.H.M. and by NIH postdoctoral fellowship GM05450 to F.J.S. *Present address: Department of Biochemistry, University of Missouri, Columbia, Missouri 65211. REFERENCES 1 2 3
4 5 6 7
8 9 10 11 12
Seidman, J.G., Barrell, B.G., and McClain, W.H. (1975) 733-760. Guthrie, C. (1975) J. Mol. Biol. 95, 529-547. Schmidt, F.J., Seidman, J.G., and Bock, R.M. (1976) J. 2440-2445. Schmidt, F.J. (1975) J. Biol. Chem. 250, 8399-8403. McClain, W.H., Seidman, J.G., and Schmidt, F.J. (1978) 519-536. Seidman, J.G., Schmidt, F.J., Foss, K., McClain, W.H. 400. Maisurian,
A.N.,
and
Buyanovskaya,
E.A.
(1973) Mol.
J. Mol. Biol. 99,
Biol. Chem. 251, J. Mol. Biol. 119,
(1975) Cell 5, 389-
Gen.
Genet.
120,
227-
229. Carre, D., and Chapeville, F. (1974) Biochemie 56, 1451-1457. Singer, M.F. (1966) in Procedures in Nucleic Acid Research, G.L. Cantoni and D.R. Davies, Eds. Vol. 1, pp. 192-202. Robertson, H.D., Altman, S., and Smith, J.D. (1972) J. Biol. Chem. 247, 5243-5251. Bikoff, E.K., Larue, B.F., and Gefter, M.L. (1975) J. Biol. Chem. 250, 6458-6255. Ghosh, R.K., and Deutscher, M.P. (1978) J. Biol. Chem. 253, 997-1000.
4139
Nucleic Acids Research
An Escherichia coli ribonuclease which removes an extra nucleotide from a biosynthetic intennediate of bacteriophage T4 proline transfer RNA
Francis J.Sclunidt* and William H.McClain Department of Bacteriology, University of Wisconsin, Madison, WI 53706, USA Received 7 August 1978 ABSTRACT The biosynthesis of bacteriophage T4 tRNA Pr, tRNA Ser, and tRNAIle requires enzymatic removal of extra nucleotides from the 3' terminus of the respective precursor RNAs. A ribonuclease activity capable of catalyzing such reactions has been partially purified from uninfected Escherichia coli using an artificial precursor RNA as substrate. A number of ribonuclease activities were resolved during purification. Use of E. coli strain BN, a mutant known to be deficient in the relevant ribonuclease activity, permitted us to identify it in wild-type cells. This activity was designated the BN ribonuclease. BN ribonuclease had an apparent molecular weight of 35,000 as measured by Sephadex gel filtration. Mg2+ was required for activity, which was optimal at [Mg2+] of 2mM. Activity did not require monovalent cations K+ or Na+. BN ribonuclease was less efficient at removing extra residues in the biosynthesis of tRNASer and tRNAIle than in the biosynthesis of tRNAPro.
INTRODUCTION
Bacteriophage T4 codes for eight transfer RNA species. These tRNAs have furnished a useful system for the study of tRNA biosynthetic pathways and of the enzymes involved in these pathways. Three host enzymes required for T4 tRNA biosynthesis have been identified (1-6). Ribonuclease P cleavages generate the 5' ends of all mature T4 tRNAs. Transfer RNA nucleotidyltransferase (E.C. 2.7.7.25)synthesizes 3' terminal C-C-A sequences of T4 tRNA P tRNA , tRNA , and tRNA . Both enzymes have been partially purified from uninfected E. coli and shown to catalyze the appropriate reactions in vitro. A third host enzyme is required for biosynthesis of T4 tRNA , tRNA Ile . Extra nucleotides are removed from the 3' end of these tRNA and tRNA sequences before the C-C-A terminus is synthesized by tRNA nucleotidyltransferase. Removal of these residues does not occur in E. coli strain BN, and this deficiency has been attributed to loss of a ribonuclease activity (6, 7). In this communication, we report the partial purification and initial characterization of this ribonuclease, termed the BN ribonuclease. G Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
4129
Nucleic Acids Research MATERIALS AND METHODS Strains. Bacteriophage strains T4D and T2L as well as Escherichia coli strains B and BN were from our collection. Materials. Chromatographic resins were obtained from Whatman Products (DE52 DEAE cellulose) and Pharmacia, Inc. (Sephadex G25 and G100). [5- H]UTP (s.a. 20 ci/mmole) was obtained as the tetrasodium salt from New England Nuclear Corporation. Venom phosphodiesterase was from P-L Biochemicals. All other reagents were commercial analytical grade reagents. RNA sequence analysis. This was performed as described previously (1). Construction of tRNA-CU*. This analogue of immature tRNA was prepared by the procedure as outlined in Fig. 1 (8). Unfractionated E. coli tRNA (235 A260 units) in 0.55 ml of 0.04 M glycine-NaOH,pH 8.7,0.01 M Mg-acetate was equilibrated at 200, then venom exonuclease (0.1 mg in 0.1 ml of the above buffer) was added and the reaction allowed to proceed at 20° for 3 hrs. Digestion was terminated by phenol extraction of the reaction mixture and the product ("tRNA-C") was precipitated at -20° by addition of 25p4 SM K-acetate, pH5.0, and 2 ml absolute ethanol. Yield: 170 A260 units tRNA-C. A partially purified preparation of tRNA nucleotidyltransferase was used to add a single *5'UMP residue to tRNA-C. The reaction mixture contained in a total volume of 4.0 ml: 200 pmole Tris Cl, pH8.5, 80 pmole Mg-acetate, 60 pmole and S-mercaptoethanol, 100 pCi (Snmole) [5units tRNA nucleotidyltransferase. (One unit catalyzes the addition of 1 pmol/min of ATP to tRNA-C.) Reaction was at 370 for 3 hrs at which time the product tRNA containing [3H]UMP ("tRNA-CU*") was isolated by phenol extraction and chromatography on DE23 cellulose (4). Assay of BN RNase. The reaction mixture contained 1-3x10 cpm of tRNACU* and enzyme in 0.1 ml Buffer A (0.OlM Tris base/0.1 M NH 4Cl/0.02 M MgCl2/ 0.001 M dithiotheitol; this solution is self-buffering at pH 8.0). At the end of the incubation period, tRNA was precipitated by addition of serum albumin and TCA (8); then the precipitate was collected on Whatman GFA glass fiber filters, washed and counted. Alternatively an ethanol-salt mixture was used to precipitate the RNA (9) and the supernatant liquid was counted. Reactions of RNase with [ 32P immature tRNAPro were done as described previ-
3HUTP,
3x104
ously (6). Partial purification of BN RNase.
Frozen E. coli B
cells, 9.0 g,
were
partially thawed in a prechilled mortar on ice. The cell paste was mixed with 18.0 g levigated alumina and ground until a smooth paste was formed. Then Buffer A (9.0 ml) was mixed with the slurry and 100 vg DNase (Worthing4130
Nucleic Acids Research ton DPFF, RNase-free) was added. The mortar was kept on ice for 40 min. The slurry was centrifuged at 8000 xg for 19 min and the supernatant removed and centrifuged again at 30,000xg for 40 min. To this S30 supernatant (6.0 ml) was added 3.0 ml of 5.0% (w/v) protamine sulfate. The slurry was stirred on ice for 15 min and then centrifuged at 30,000xg for 30 min. To the resulting supernatant (8.2 ml) was added 2.45 g (NH4)2S04 (Mann, enzyme grade). The precipitate from this step was isolated by centrifugation and redissolved by addition of 1.5 ml Buffer A. This solution was divided into two aliquots which were desalted on a column of Sephadex G25 (0.9 cm x 29 cm in Buffer A). The combined desalted enzyme fractions were applied to a column (0.9 cm x 14 cm) of Whatman DE52 cellulose' in Buffer A. The column was washed with two column volumes of Buffer A and then with a linear gradient of 75 ml each of Buffer A and Buffer B (Buffer B is the same as Buffer A except that it also contains 0.2 M KC1). The peak enzyme fraction which eluted from DEAE cellulose beginning at 14 mmho or approximately 0.1 M KC1 (the second peak in Fig. 3, top) was further fractionated on Sephadex G200. Two ml of the DE52 fraction were applied to and eluted from a column of Sephadex G200 (0.9 cm x 60 cm) in Buffer A. Fractions of 1.0 ml were taken. RESULTS
Assay of BN Ribonuclease. In our previous work (6) this RNase activity was detected in crude extracts of E. coli by its ability to alter the 3' terminus of immature T4 tRNA ro, and the reaction was followed by fingerprint assay. Since fingerprint assays are expensive and time consuming,
they are unsuitable for use in enzyme purification. We therefore developed a rapid and simple assay using an artificial substrate (Fig. 1). This method utilized several reactions of tRNA, characterized by Carre and Chapeville (8), to produce an analogue of immature tRNA . First, treatment of bulk E. coli tRNA with venom exonuclease at low temperature removed the terminal C-A sequences from the tRNA chains. The resulting tRNA-C was a substrate for tRNA nucleotidyltransferase which in the absence of ATP and CTP added a single [5-3H]UMP (8). The product of the reaction (tRNA-CU*) was a modified tRNA ending C-U* instead of C-C-A, where the U* denotes a terminal [3H] uridine nucleoside. Because of its resemblance to immature (which ends C-U), we reasoned that tRNA-CU* might tRNA be a substrate for the processing RNase deficient in E. coli strain BN. The results in Fig. 2 show that an extract of wild-type E. coli B was 4131
Nucleic Acids Research venom tRN A-CCA OH tRNA-CCAOH
exonucleose tRNA-COH ~20 tRNA
nucleotidyl -fH] UT P
UMP+ *tRNACOH Fig. 1.
~~~BNRNose -
t RNA- CUOH
Scheme for assay of the BNl RNase.
100
Ea. 80
'Z soIa.
20 60-
E. coil BN
C e
40-
0
E. coil B
20
20
40
60
Time, min Fig. 2. Release of [3H] from tRNA-CU* by cell-free extracts. concentration in reaction mixtures was 10 mg/ml.
Protein
about twice as efficient in removing [5- H]U from the artificial substrate as This result meant that the artificial substrate could be used to assay the relevant ribonuclease. However, tRNA-CU* was a substrate for other nucleases since vig. 2 shows that some of the [3H]U was rendered acid soluble by the mutant extract. Partial purification of BN RNase. Throughout the purification of this enzyme, initial fractionation steps were assayed using tRNA-CU* as the substrate. Active fractions were then examined for ability to remove pU nucleotide from [ P]-labeled immature tRNA . The latter step was necessary because several cellular nucleases removed [ H]U from tRNA-CU*.
was an extract of E. coli BN.
4132
Nucleic Acids Research BN RNase activity did not associate with ribosomes when extracts were The activity could be purified prepared in the presence of 0.1 M NH4Cl. from either an IQ00 supernatant fraction or from the supernatant remaining after protamine sulfate precipitation of ribosomes and large nucleic acids. Ammonium sulfate fractionation experiments indicated that all BN RNase activ-
ity was precipitated by addition of 0.3 g/ml of (NH4)2S04 to either the S100 fraction or to the supernatant rema-ining after protamine sulfate precipitation.
The (NH4)2S04 precipitate was resuspended in Buffer A and desalted on Sephadex G25. The fraction was then applied to a column of Whatman DE52 DEAE cellulose. Elution was with a linear KC1 gradient (0-0.2 M) in Buffer A. Fig. 3 (top) shows that the majority of the wild-type (E. coli B) RNase activity was eluted from the column in two incompletely separated peaks. The RNase activity from E. coli BN, in contrast, eluted in only one peak (Fig. 3,
bottom), at the position of the first main peak in wild-type E. coli B. The second peak of wild-type RNase activity therefore probably corresponds to the RNase which is deficient in E. coli BN. In control experiments (data not shown) this second peak of wild-type activity removed a pU nucleotide unit
from
, while the residual RNase activity from P-labeled immature tRNA E. coli BN eluting at the same conductivity (beginning at 14 mmho) was incapable of removing this pU nucleotide. BN RNase was further separated from contaminating RNase activity by chromatography on Sephadex G200, as shown in Fig. 4. This procedure also
indicated that the molecular weight of BN RNase is approximately 35,000. BN RNase at this state of purification removed a single pU residue from Pro . Data allowing this conclusion are shown in Fig. 5, where immature tRNA the RNase A (pyrimidine-specific) fingerprints of immature tRNAPrO before and after BN RNase treatment are presented. The 3' terminal sequence of immature is G-G-A-G-A-C-U. RNase A digestion of tRNA terminating in this tRNA sequence gives G-G-A-G-A-Cp as the 3' terminal product (see Fig. 5a). (The other product from the 3' terminus of the tRNA, uridine, does not contain frac132pI phosphate and so is not observed by autoradiography.) The G200 Pro leavtion of BN RNase removed the terminal pU residue from immature tRNA ing a new 3' terminus, G-G-A-G-A-C; the position of this oligonucleotide in the RNase A fingerprint is shown in Fig. Sb. Fig. 6 shows how the sequence of G-G-A-G-A-C was characterized. Comparison of the amount of 3' terminus removed from immature tRNAPr by crude cell-free extracts (see Seidman, et al (6)) with that removed by BN RNase purified through G200 chromatography
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A2-
0
cpm on
1200 E. coli BN
0.6
30-
600
A280 800
I 8004
20-
Conductivity, 10
cpm on filter AAA
mmho
A~~~~~ 0.2
10
10A 20
30
40
1200
FRACTION NUMBER Fig. 3. tions.
Chromatographic profiles on DE52 cellulose of the
(NH4)2S04
frac-
indicated an overall enrichment of between 50- and 100-fold. and The G200 fraction of BN RNase was incubated with immature tRNA the reaction mixture was spotted onto DEAE paper for electrophoresis at pH 3.5. Small products liberated in this reaction included all four 5' mononucleotides and short oligonucleotides 2-3 residues in length. The same products were found in greater yield when the peak of RNase activity eluting before BN RNase was examined. This activity may be RNase D, an enzyme of 60,000 molecular weight that apparently generates 5' mononucleotides from 4134
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0.2 0
A280
CD
0.1
41
-J
0
FRACTION NUMBER Fig. 4. Sephadex G200 chromatography of the DE52 fraction. The bir graph represents the molar yield of G-G-A-G-A-C following treatment of [ 2p] immature tRNAPro with 0.2 ml column fraction containing BN RNase (see Fig. 5).
tRNA chains (12).
It is possible that RNase D or a similar activity contami-
nates the BN RNase fraction as a result of incomplete column resolution. Al-
ternatively, the BN RNase may have a broad specificity. Further purification is needed to resolve this uncertainty. Properties of purified BN RNase. BN RNase purified through the Sephadex G200 step was exchanged with Buffer A lacking Mg by Sephadex G25 chromaof was addition reconstituted tography. Activity by differing concentrations of MgCl2 to the enzyme. The BN RNase fractions containing various concenion were assayed by reacting equal amounts of enzyme with trations of Mg 132pI immature tRNAPro and fingerprinting the RNA products. These experifor its activity (Fig. 7); maximal ments revealed that BN RNase requires Mg 2+ of 2mM. At concentrations above this level, activity was obtained at a [Mg 4135
Nucleic Acids Research a
I.h 46
40.-.
_Al
*%-CG,4,(--4Cp 1%.
G(,A 7AC0
.-.
2nd
I
0
'*'V
GG
AC.
*S
9
a
b
Fig. 5. RNase A fingerprint of immature treatment with purified BN RNase.
tRNAro
(a) before and (b) after
G-G -A-G-A-C
RNase T2 RN*eT Gp(3), Ap(2) Venom
pG(2), pA(2), pC
RNasw RNo- TiT1
Gp(2), RNose T2
A-Gp,
A-C
RNose T2 Gp
RNase T2 Ap, Gp
-
Venom
Fig. 6.
Ap
pC
Characterization of the new 5' terminal sequence, G-G-A-G-A-C.
markedly inhibited the enzyme so that the activity was reduced two2 fold at [Mg ] of 10 mM, and was barely detectable at 20 mM Mg Monovalent cations were apparently not required for activity. The activity of BN RNase as measured in fingerprint assays was not affected by exchange on Sephadex G25 with 0.01 M TrisCl, pH 8.0/0.002 M MgCl2 buffer concontaining either KC1, NaCl or no monovalent cation. However, the enzyme may
Mg
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a
w
z 0.2
0
10-4
i0-3
2
[Mg2] M Fig. 7. Effect of [Mg 2+] on BN RNase activity as measured by the yield of G-G-A-G-A-C. Reaction mixtures contained 25 ,ug protein. have carried along a required cation from Buffer A to allow the reaction to proceed. Exhaustive dialysis of purified BN RNase against buffer A was not possible since activity was lost. Purif ied BN RNase activity was somewhat unstable at 0° C ., with a half-
life of 5-7 days. The enzyme could, however, be stored-frozen at -70° for at least one month without loss of activity. Reduced reaction with other potential substrates. Besides tRNA Potwo other bacteriophage T4 tRNA species require the removal of extra nucleotides prior to C-C-A synthesis . We isolated the appropriate [ 32p'L] labeled precursors and subj ected them to reaction under conditions which proved optimal .Reaction with these substrates was for removal of pU from immature tRNA less ef ficient . The tRNAPo-tRNA Srprecursor RNA requires removal of U-A-A from the 31' end of the molecule. The 3 ' terminal sequence of precursor RNA is C-C-U-C-CG-U-A-A; removal of pU-A-A in vivo generates a precursor RNA which ends C-c-C U-C-C-G (1). This reaction does not occur in E. coli strain BN (6). The reaction of BN RNase (purif ied through the DE52 column step) with precursor RNA was less complete than that with immature tRNA tRNA -
4137
Nucleic Acids Research tRNA
, since the molar yield of C-C-U-C-C-G was estimated to be 0.01-0.03;
resulted in a yield of G-G-A-G-A-C parallel reaction with immature tRNA equal to 0.5. Characterization of C-C-U-C-C-G included its position on the two-dimensional fingerprint (6) and the products of RNase T2 digestion. Similar results were obtained when BN RNase was reacted with T4 tRNA Ile Ile . The latter RNA species is pretRNA precursor RNA or immature tRNA P cleavage of the precursor RNA, though it has not sumably derived by RNase been determined if this cleavage precedes conversion of 3' U-A-U residues to C-C-A. Both of these RNAs terminate with U-C-A-U-A-U. In vivo, the BN RNase is required for removal of pU-A-U from the 3' end of the tRNA e sequence (6). When either RNA species was treated with BN RNase purified through the DE52 column step, removal of pU-A-U residues occurred to a limited extent (0.020.03 molar yield of U-C-A). The product U-C-A was identified solely by its position on the two-dimensional RNase T1 fingerprint.
DISCUSSION In the present work we have partially purified an enzyme, the BN RNase, Pro . This which removes a pU residue from the 3' terminus of immature tRNA reaction occurs in vivo in wild-type cells but not in mutant strain BN (6). As expected, BN RNase was not detected in strain BN. While purified BN RNase acted quite efficiently on immature tRNA
ro, it
was less efficient with immature tRNA Ile, the tRNA Pr-tRNASer precursor RNA
and the tRNA hr-tRNA precursor RNA. Removal of 3' nucleotides from the latter group of RNAs is also abolished by the BN mutation in E. coli (6). If the BN phenotype is the result of a single mutation, purified BN RNase should catalyze removal of 3' residues from all these RNA species. Several explanations for reduced efficiency can be mentioned. This reduced efficiency may result from a greater complexity in removing three nucleotides as opposed to Pro . If the probability of removing pU one nucleotide from immature tRNA is 0.5, then the probability of removing three resifrom immature tRNA 3 dues might be (0.5) =0.125. Alternatively, the BN RNase was purified from
uninfected E. coli; bacteriophage induced modification of the enzyme may occur. Finally, it is possible that the in vivo state of BN RNase is important for its action. Assay methods for processing ribonucleases of tRNA precursors have previously relied on two types of measurements, either structural changes (e.g. cleavage) of isolated precursor RNAs (10) or the production of a functional tRNA product which can itself be measured (11). This paper presents 4138
Nucleic Acids Research third approach which, because of its relative simplicity,
a
for
a
variety of
enzymes
may prove
useful
of this type.
ACKNOWLEDGEMENTS We thank Prof. Robert M. Bock for suggesting the
substrate in assaying the work
was
enzyme
use
of
an
artificial
and for other helpful discussions.
This
supported by NIH grant AI10257 and NIH Research Career Development
Award AI10002 to W.H.M. and by NIH postdoctoral fellowship GM05450 to F.J.S. *Present address: Department of Biochemistry, University of Missouri, Columbia, Missouri 65211. REFERENCES 1 2 3
4 5 6 7
8 9 10 11 12
Seidman, J.G., Barrell, B.G., and McClain, W.H. (1975) 733-760. Guthrie, C. (1975) J. Mol. Biol. 95, 529-547. Schmidt, F.J., Seidman, J.G., and Bock, R.M. (1976) J. 2440-2445. Schmidt, F.J. (1975) J. Biol. Chem. 250, 8399-8403. McClain, W.H., Seidman, J.G., and Schmidt, F.J. (1978) 519-536. Seidman, J.G., Schmidt, F.J., Foss, K., McClain, W.H. 400. Maisurian,
A.N.,
and
Buyanovskaya,
E.A.
(1973) Mol.
J. Mol. Biol. 99,
Biol. Chem. 251, J. Mol. Biol. 119,
(1975) Cell 5, 389-
Gen.
Genet.
120,
227-
229. Carre, D., and Chapeville, F. (1974) Biochemie 56, 1451-1457. Singer, M.F. (1966) in Procedures in Nucleic Acid Research, G.L. Cantoni and D.R. Davies, Eds. Vol. 1, pp. 192-202. Robertson, H.D., Altman, S., and Smith, J.D. (1972) J. Biol. Chem. 247, 5243-5251. Bikoff, E.K., Larue, B.F., and Gefter, M.L. (1975) J. Biol. Chem. 250, 6458-6255. Ghosh, R.K., and Deutscher, M.P. (1978) J. Biol. Chem. 253, 997-1000.
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