the journal of no. 50, of 16, printed in u.s.a. … · substrate specificity of an rnase 111-like...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 50, Issue of December 16, pp. 3145041456, 1994 Printed in U.S.A.

Substrate Specificity of an RNase 111-like Activity from Bacillus subtilis*

(Received for publication, August 11, 1994, and in revised form, October 11, 1994)

Sutapa Mitra and David H. BechhoferS From the Department of Biochemistry, Mount Sinai School of Medicine of City University New York, New York, New York 10029-6574

Bacillus subtilis bacteriophage SP82 codes for several early RNAs that were shown previously to be cleaved by an RNase 111-like enzyme called “Bs-RNase 111.” Cloning of DNA fragments that encode these RNA sequences downstream of a T7 RNA polymerase promoter allowed the synthesis of substrates that were used to test the cleavage specificity of Bs-RNase 111, which was purified from a protease-deficient strain of B. subtilis. Single nucleotide changes at or near the cleavage site and deletions upstream and downstream of the cleavage site were constructed. The effects of these changes on the rate of Bs-RNase I11 cleavage were measured. The activity of Bs-RNase I11 and Escherichia coli RNase I11 on heterologous substrates was also tested. Although the local environment of the site of Bs-RNase I11 cleavage appears very similar to that of E. coli RNase 111, there are important differences in their substrate specificity.

Bacterial endoribonucleases are involved in the processing of bacteriophage and ribosomal RNAs and the initiation of decay of several messenger RNAs. The most well characterized of these endonucleases is ribonuclease I11 (RNase 111) of Escherichia coli, which cleaves RNAs containing specific double-stranded structures. Ribosomal RNA operon transcripts, early RNAs of bacteriophages T7 and T4, and the PL transcript of bacteriophage h were the first to be identified as in vivo substrates of RNase I11 (1). More recently, i t has been found that certain bacterial messenger RNAs are also cleaved by RNase 111, and this can affect mRNA half-life and the level of gene expression (see Ref. 2 for a recent review). Expression of certain h phage genes are affected by RNase I11 binding in the absence of cleavage (3) or by the cleavage of a double-stranded structure formed by complementary RNAs (4). Small, in vitro transcribed RNA substrates have been used recently to define more precisely the recognition elements that direct RNase I11 cleavage (5-7).

An enzyme activity similar to that of RNase 111 was found in Bacillus subtilis by Panganiban and Whiteley (8) and was called Bs-RNase 111’ (for B. subtilis RNase 111). It was shown that purified Bs-RNase 111 cleaved early transcripts of the B. subtilis bacteriophage SP82 (8), as well as B. subtilis precursor

* This work was supported by United States Public Health Service grants GM-39516 and GM-48804 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed: Box 1020, Dept. of Biochemistry, Mount Sinai School of Medicine, 1 Gustave Levy Place, New York, NY 10029-6574. Tel.: 212-241-5628; Fax: 212-996-7214; E-mail: [email protected].

The abbreviations used are: Bs-RNase 111, RNase I11 from B. subtilis; PMSF, phenylmethylsulfonyl fluoride; PCR, polymerase chain reaction; nt, nucleotide(s).

rRNA(9). The E. coli and B. subtilis RNase I11 enzymes differed in their sensitivity to monovalent cation concentrations, and it was reported (9) that they could cleave only homologous substrates (i.e. RNase 111 of E. coli could not cleave B. subtilis rRNA or SP82 early mRNA, and Bs-RNase I11 could not cleave E. coli rRNAor T7 mRNA). The SP82 RNAstructures that were cleaved by Bs-RNase I11 resembled the T7 RNA substrates in that they comprised a stem-loop region interrupted by an internal loop. However, Bs-RNase I11 cleavage sites on three SP82 RNAs were mapped to the upper loop region (81, quite different from the mapped cleavage sites of E. coli RNase I11 on T7 early RNAs, which are in or close to the internal loop sequence (10). Previously, we cloned an SP82 DNA fragment encoding the Bs-RNase I11 ”A” cleavage region into the coding sequence of the ermC gene. A single cleavage site was mapped in the internal loop sequence, at a location similar to that of the RNase I11 cleavage site of T7 early RNAs (11).

Since little is known about the regulation of gene expression at the level of RNA processing in B. subtilis, we undertook a study of the B. subtilis version of RNase 111, with the long range goal of determining its relevance to the initiation of mRNA decay and control of gene expression in B. subtilis. The current report describes an initial characterization of the cleavage specificity of Bs-RNase 111. Various in vitro transcribed, labeled RNAs were treated with purified Bs-RNase I11 to determine the cleavage site and the effect of changes in the RNA sequence on the ra te of cleavage.

EXPERIMENTAL PROCEDURES Bacterial Strains-The B. subtilis extract was prepared from the

protease-deficient strain GP208, provided by Dr. A. Sloma, which is metC amyE Aapr Anpr Aisp-1. E. coli strain DH5a was the host for plasmid pJFD4 and its derivatives.

Purification ofBs-RNase III-Bs-RNase I11 was purified as described by Panganiban and Whiteley (9), with some modifications. Cultures of B. subtilis strain GP208 were grown i n l ” medium (25% Tryptone, 2% yeast extract, 0.3% %HPO,, 3% glucose) at 37 “C and harvested at late log phase (270-300 Klett units). Cultures were cooled to less than 10 “C and then centrifuged at 3,000 x g for 10 min. Pellets were washed once with 10 m~ Tris acetate, pH 8.0, 14 mM Mg(AcO),, 60 m~ potassium acetate, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride (PMSF). Cell pellets (approximately 6 g/liter of culture) were stored at -70 “C. Sonication, ammonium sulfate precipitation, and phenyl- Sepharose chromatography were as described elsewhere (9). The phenyl-Sepharose column was washed with buffer C (10 mM Tris, pH 7.5, 10% glycerol, 1 mM EDTA, 0.5 mM dithiothreitol, 20 mg/liter PMSF, and 20 mg/liter I-tosylamide-2-phenylethylchloromethyl ketone containing decreasing amounts of ammonium sulfate from 2.0 M to no ammonium sulfate, as described. Fractions from the last wash containing the great- est amounts of activity were pooled, concentrated by ultrafiltration through a Centriprep-IO membrane (Amicon), and diluted with buffer D (buffer C with the concentrations of PMSF and 1-tosylamide-2-phenylethylchloromethyl ketone reduced to 10 mg/liter) containing 2.0 M ammonium sulfate to give a conductivity equal to that of buffer D containing 0.5 M KC]. The preparation was mixed in a 15 ml centrifuge tube with 0.1 ml of poly(I).poly(C) agarose, which was equilibrated with buffer D containing 0.5 M KCl. The mixture was shaken overnight on a

31450

Endoribonuclease of B. subtilis 3 145 1

Purification of Bs-RNase 111 TABLE I

Fraction Total protein Total activity Specific activity Yield Purification ~~~~~~~ ~ ~

rng units’ unitslrng % -fold 5-30 3298 5.9 x 106 1,789 100 1 65% ammonium 509 2.7 x lo6 5,305 46 2.97

Phenyl-Sepharose 97 6.5 x 105 6,701 11 3.75 Poly(I).poly(C) agarose 0.0019 7.2 x 103 3,789,474 0.12 2118

sulfate precipitate

a A unit is defined as the amount of activity that gives 50% cleavage of pJFD4 XbaI RNA in 5 min at 37 “C under standard assay conditions.

nutator at 4 “C to allow maximum binding of the activity. After incubation the mixture was centrifuged at low speed and the supernatant was carefully removed. The resin was washed twice with 1 ml each of buffer D containing half molar increments of NaCl from 0.5 to 2.5 M. The purified Bs-RNase I11 shown in Fig. 1, lane 2, eluted in the second 1.0 M wash. This fraction and the fraction from the first 1.5 M wash were pooled and dialyzed against 1 liter of 10 mM Tris, pH 7.9, 1 mM EDTA, 0.2 M NaCl, 10% glycerol, 0.5 M dithiothreitol. The dialyzed enzyme was stored in 0.02-ml aliquots a t -20 “C in the presence of 50% glycerol and 0.1 mg/ml bovine serum albumin to minimize the loss of activity. Protein concentrations were determined using the MicroBCA protein assay re- agent (Pierce). The protein gel in Fig. 1 was a 12% polyacrylamide gel according to Laemmli (12) and was stained with silver nitrate (13). The amount of protein in lane 2 of Fig. 1 was 108 ng, which was determined by densitometry of protein standards run on the same gel containing between 50 and 500 ng of proteidane (not shown).

Plasmids-Plasmid pJFD4 (Fig. 2 A ) , which contained the “A” cleavage region of SP82 early RNA, was constructed previously in this lab- oratory (11) and is described in the text. To clone the “ B and “C” cleavage regions, pairs of PCR primers with BamHI cleavage sites at the ends were made that contained either the SP82 nucleotides (nt) 674-689 and the sequence complementary to n t 786-800 (for the B region), or SP82 nt 1228-1242 and the sequence complementary to n t 1333-1348 (for the C region). Numbering of the SP82 sequence is from Panganiban and Whiteley (8). PCR amplification products were digested with BamHI and cloned into the BamHI site of pGEM-3Zf(+) (Promega). Plasmid pKH5 was a deletion derivative of pJFD4 and was constructed by deleting the small SmaI-ScaI fragment (Fig. 2.4) and religating the large SmaI-ScaI fragment with the ori-containing ScaI fragment. Bal-31 deletion mutations were constructed by linearizing pJFD4 at the HpaI site and digesting with Bal-31 nuclease for short times (10-s time points). The Bal-31-treated DNA was religated and used to transform E. coli strain DH5a. Plasmids that were missing the HpaI site and had small deletions were selected for sequencing.

For construction of point mutations at the cleavage site, an M13 derivative was made on which both mutagenesis and subsequent transcription reactions could be performed. The SP82 insert in pJFD4 was first cloned into the T7 promoter vector pIBI31 (IBI/Kodak Scientific Imaging Systems), which has an EcoRV restriction endonuclease site upstream of the T7 promoter sequence. The T7 promoter-SP82 DNA was cloned on an EcoRV-XbaI fragment into SmaI + XbaI-digested M13mp19, yielding the M13 derivative designated EG153. This provided a single-stranded template for mutagenesis by the procedure of Kunkel et al. (14) Replicative forms were linearized and transcribed to give RNA substrates with single nucleotide changes at the cleavage site. EG153 was also used to create EG200, which contained a deletion of the first 148 n t of the SP82 sequence originally cloned into pJFD4. The change of n t 219 from a G to a T was done by the method of Lai et al. (15).

T7 R1.1 RNA was transcribed from NciI-digested plasmid pAR1450 (kindly provided by Dr. A. Nicholson) to give a 131-nt RNA (16).

Danscription Reactions-Transcription reactions were performed as detailed in the Promega Protocols and Applications booklet. T7 RNA polymerase was purchased from New England Biolabs; radiolabeled nucleotides were purchased from DuPont-NEN. Uniform labeling was with [a-32PlUTP. The major transcription product was isolated from a 5% denaturing polyacrylamide gel and recovered as described elsewhere (17). The amount of RNA recovered was determined by scintil- lation counting, comparing the specific activity of the input label with the countslmin in the final recovered product. Reverse transcriptase reactions were performed as previously described (18), using as primer an oligonucleotide complementary to bases beginning 25 n t downstream of the Bs-RNase I11 cleavage site.

Bs-RNase IIIAssay-The assay for Bs-RNase I11 activity was a modification of the one described by Panganiban and Whiteley (9). The

standard assay mixture contained 10 mM Tris-HC1, pH 7.9,3 mM MgCl,, 100 mM NaCl, 0.04 pmol of labeled RNA, and 0.8 ng of purified enzymd60 pl of assay volume. Reaction mixtures were prewarmed a t 37 “C for 2 min, and the reaction was initiated by the addition of enzyme. The “zero” time point in the experiments described in the text was the time elapsed from the addition of enzyme until removal of the first aliquot (about 5 8). The reaction was terminated by removal of a 10-pl aliquot to 5 pl of stop solution. Samples were heated at 80 “C for 2 min and cooled on ice. The products of the reaction were analyzed by urea- polyacrylamide gel electrophoresis followed by autoradiography. Radio- activity in the bands was quantitated using a PhosphorImager instru- ment (Molecular Dynamics). Determination of the cleavage rates for the mutant substrates was done at least twice for each mutation.

The assay buffer for E. coli RNase I11 was 30 lll~ Tris-HC1, pH 8.0, 160 mM KCl, 5 mM spermidine, 0.1 mM dithiothreitol, 5 mM MgCl,. For the experiment in Fig. 7, 28.5 units of E. coli RNase I11 were used per reaction.

RESULTS

Purification of Bs-RNase IIZ-Table I summarizes the fold purification of Bs-RNase I11 through a modification of the origi- nal procedure (see “Experimental Procedures”). Greater than a 2,000-fold purification was achieved but with a low yield of activity (0.12%). The most purified fraction from the poly(I).poly(C) agarose step was electrophoresed on a 12% denaturing polyacrylamide gel and the gel was silver stained (Fig. 1). The fractions from the 1.0 M NaCl wash (lane 2) and the 1.5 M NaCl wash, which also contained a single detectable band but with less protein (not shown), were pooled and used to charac- terize Bs-RNase I11 cleavage activity.

Cleavage of the SP82 A Site-The parent plasmid molecule that was used for in vitro transcriptions is shown in Fig. 2 A . Plasmid pJFD4 contained a 373-base pair fragment of PCR- amplified SP82 DNA that was cloned into the BanHI site of pGEM-BZA+), as described previously (11). Runoff transcription of this fragment from the T7 RNA polymerase promoter gave an RNA molecule that contained a site for cleavage in vivo (the “ A site of Ref. 8) and for purified Bs-RNase I11 (9). The RNAFOLD program of Zuker and Stiegler (19) was used to predict a putative secondary structure for the region of SP82 sequence in which cleavage was shown to occur (Fig. 2 B ) .

Plasmid pJFD4 DNA was linearized with XbaI, which cuts immediately downstream of the distal BamHI site, and transcription was carried out with T7 RNA polymerase in the presence of w3?-labeled UTP. The resultant uniformly labeled, 403-nt transcript (“pJFD4 XbaI RNA”) was cleaved at a single site when incubated in the presence of purified Bs-RNase I11 (Fig. 3A). We had previously mapped the in vivo cleavage site to the U-G bond at nucleotides 215-216 in Fig. 2 B . Prominent bands representing 5’- and 3”cleavage products were evident (labeled 5’ and 3’ in Fig. 3A), with the predicted sizes of 240 and 163 nt, respectively. Cleavage of pJFD4 XbaI RNA was taken as the standard cleavage reaction, and the activity of Bs-RNase I11 on all other substrates was compared to that of pJFD4 XbaI RNA. Under the standard assay conditions (see “Experimental Procedures”), 97% of the substrate was cleaved by 8 min.

31452 Endoribonuclease of B. subtilis

1 2 3 - ”” . -

46

30

21.5

’ 14.3

FIG. 1. Polyacrylamide gel electrophoresis of purified protein fractions. Lane 1, phenyl-Sepharose fraction; lane 2, 1.0 M wash fraction from poly(I).poly(C) agarose; lane 3, protein standards (molecular masses given in kilodaltons at right).

Cleavage of the SP82 B and C Sites-In addition to the A site described above, two other SP82 early RNAs contain cleavage sites, and these were designated the “B“ and “C” sites (8). The predicted secondary structures of the RNA containing these regions are shown in Fig. 4. The overall shape and stability of these predicted structures were similar to that of the A site (Fig. 2B). SP82 DNA encoding the B and C cleavage regions was amplified by PCR and cloned as BamHI fragments into pGEM-SZf(+) (see “Experimental Procedures”). As part of a study concerned with the ability of SP82 RNA sequences to confer stability on downstream mRNAs, the in vivo cleavage sites for these RNAs in B. subtilis were determined.’ These were mapped to the sites shown by the arrows in Fig. 4. The cleavage site in the B and C regions was between U and G residues in or close to the internal loop region, as was the case for the A site cleavage. In vitro transcribed RNAs containing the B and C cleavage sites were prepared and incubated with purified Bs-RNase I11 (Fig. 3). The sizes of the observed cleavage products were the expected sizes if cleavage in vitro was occurring at the same site as the site mapped in vivo. However, when compared to cleavage of theA site (pJFD4XbaI RNA), the rate of cleavage was about 4-fold slower for both the B and C sites (Fig. 5).

Length Requirements for Cleavage-To define more precisely the RNA sequence that was required for recognition and cleavage by Bs-RNase 111, shorter RNA substrates containing the A site were prepared. A HpaI restriction endonuclease site was located 26 n t downstream of the stem structure (Fig. 2 B ) . Plas- mid pJFD4 DNA was linearized with HpaI and transcribed with T7 RNA polymerase. The resultant transcript (pJFD4 HpaI RNA) was also cleaved rapidly when incubated with Bs- RNase 111. The rate of cleavage was about 2-fold slower than that of pJFD4XbaI RNA. The precise site of cleavage for pJFD4 HpaI RNA was mapped by reverse transcriptase analysis, as was done previously for the in vivo transcribed RNA that contained the SP82 A region (11). The results of the reverse transcriptase analysis (Fig. 6) showed that cleavage occurred at the same site in vivo and in vitro. Previous reverse transcriptase experiments (11) showed that the cleavage site was as indicated in Fig. 2B. The rapid cleavage of pJFD4 HpaI RNA dem-

K. K. Hue, S. D. Cohen, and D. H. Bechhofer, manuscript in preparation.

A

B

m

SP82 A site (-19.6 Kcai)

mid pJFD4. Relevant restriction sites are shown. The lighterpart ofthe FIG. 2. Template used for in vitro transcription. A, map of plas-

circle represents the 373-base pair BamHI fragment containing SP82 phage sequences. Location of the T7 RNA polymerase promoter is rep- resented by the small p , with the arrow pointing in the direction of transcription. B, map of the inserted SP82 BamHI fragment with the predicted secondary structure and stability for the sequence surrounding the Bs-RNase I11 cleavage site. Numbering is from the beginning of the inserted SP82 sequence. Arrow between nt 215 and 216 shows the Bs-RNase I11 cleavage site. Positions of relevant restriction sites are indicated; nucleotides that constitute the ScaI restriction site in the stem loop are underlined (AGTACT). The upstream deletion end points for three Bal-31 deletion mutations (A223, A228, A234) are shown. The boxed G residue a t n t 219 was changed to a U in plasmid pJFD15.

onstrated that only 39 n t of RNA downstream of the cleavage site was required for efficient cleavage by Bs-RNase 111.

To determine whether sequences upstream of the cleavage site were required for activity, the pJFD4 sequence was deleted such that transcription from the T7 promoter began at a site 24 nucleotides upstream of the beginning of the stem-loop structure shown in Fig. 2B. The template for this transcription was HpaI-digested EG200 (see “Experimental Procedures”), which gave an RNA substrate that was 106 nt long. Bs-RNase I11 cleaved EG200 HpaI RNA only 1.5-%fold slower than pJFD4 XbaI RNA. Taken together, these results show that endonucleolytic cleavage by Bs-RNase I11 is only moderately depend- ent on extended upstream or downstream sequences outside of the stem-loop structure.

Deletion Mutations-To further delineate the sequences required for recognition and cleavage, deletion mutations of the pJFD4 parent plasmid were constructed (Table 11). Adeletion of sequences upstream of the cleavage site was made that removed SP82 sequences up to the ScaI site, yielding plasmid pKH5. The ScaI site was located in the left hand side of the stem (Fig. 2 B ) , so that transcription of linearized pKH5 DNA would yield an RNA that contained the site of Bs-RNase I11 cleavage but with an altered secondary structure surrounding the cleavage site. When pKH5 DNA was linearized and transcribed, the resultant transcript was not cleaved detectably by Bs-RNase I11 (Fig. 5). This suggested that the putative second-

Endoribonuclease of B. subtilis 31453

SP82 RNAs. The three uniformly labeled FIG. 3. Bs-RNase I11 cleavage of

SP82 RNA substrates were incubated with purified Bs-RNase I11 under standard assay conditions. Aliquots were removed a t times indicated (minutes). Con- trol lane (-) had no enzyme added. FL, full-length substrate. Position of the 5'- and 3"cleavage products are shown.

U A C-G C-G U G A-U C-G U G U-A A-U U-A G-C A-U U-A

U-A- U G G-C G-C

G-C A A

G U

CA AA C-G

G ~ G u c C-G C-G U G A-U C-G C-G U G A-U U-A

G U AC :E+-

U-A C-G

U G G-C G-C A-U U-A

AU UG G U

B SITE C SITE (-1 6.4 Kcal) (-1 6.7 Kcal)

- 0 1.5 3 4.5 6 I

i A site B s ~ t e

(pJFD4 X h a l RNA)

2ool~ G A C C-G C-G C-G A-U C-G U G

A C-210 C-G

,,"U-A G-C

A C U A C

GU U-A G'\

G q - m

180-C-G G-C

A C

A-U C-G U-A C-G 230 ''Y G-C I

CCC UAC

pJFDl5 (-16.1 Kcal)

FIG. 4. Predicted secondary structures of the SP82 B and C sites and A site in plasmid pJFD15, which contained a G to U change at nt 219 (bored). The site of Bs-RNase 111 cleavage is shown by the arrow.

ary structure shown in Fig. 2B was required for Bs-RNase I11 cleavage.

To make deletions downstream of the cleavage site, Bal-31 deletion mutations of pJFD4 were constructed by linearizing pJFD4 DNA with HpaI and incubating with Bal-31 nuclease at room temperature for short time points. Fig. 2B shows the deletion end points of some of the Bal-31 deletion mutations that were isolated and sequenced. Three Bal-31 deletion plasmids (designated A223, A228, and A234, according to their 5' deletion end points) were linearized with XbaI and transcribed with T7 RNA polymerase to give RNA substrates that were incubated with Bs-RNase 111. In several experiments RNA transcribed from A223, which was deleted for one half of the lower stem, was degraded a t a slow rate when incubated for greater than 4 min in the presence of the enzyme but did not give the expected products of endonucleolytic cleavage at n t 215-216. RNA transcribed from A228, which was deleted for sequences starting at the last base of the lower stem, was cleaved at a rate approximately 20-fold lower than the wild-

C site

I 0 1 2 3 4 5 6 Time (min)

1 " F L

"5 ' "3'

I - ~"_I_

FIG. 5. Cleavage rate of SP82 RNAsubstrates. The data shown in Fig. 3 were plotted as percent (converted to log) of full-length RNA remaining uersus time of incubation. Straight lines are linear regres- sion. Symbols: 0, A region; 0, B region; M, C region; x, pKH5 (ScaI deletion). The relative rates of cleavage given in the text are from the slopes of the linear regressions.

type. RNAtranscribed from A234, whose deletion end point was 5 nt downstream of the predicted stem structure, was cleaved by Bs-RNase I11 &fold slower than the wild-type substrate. These results suggest that sequences comprising the predicted secondary structure and, perhaps, neighboring nucleotides are important in maintaining a Bs-RNase I11 recognition site.

Point Mutation in the Lower SternSince the Bal-31 deletion mutation A228 demonstrated that the nature of the lower stem was important, a change was made that was predicted to alter the structure of the lower stem without changing its size. The G residue at n t 219 was changed to a T residue, to give plasmid pJFD15 (Fig. 2 B ) . The predicted secondary structure of the relevant region of pJFD15 XbaI RNA is shown in Fig. 4. While the upper stem structure is predicted to remain unchanged, the number of nucleotides in the internal loop and the lower stem structure are predicted to be altered. pJFD15 XbaI RNA was assayed with Bs-RNase 111, and the results showed that cleavage occurred at a rate about 10-fold slower than for pJFD4XbaI RNA.

Point Mutations a t the Cleavage Site-To assess the contri- bution of the specific nucleotides at the cleavage site, the U residue at n t 215 was changed by oligonucleotide mutagenesis to each of the other three possible bases. All three mutant substrates showed substantial cleavage activity, although a t reduced rates (Table 111). RNA with the U "-f C change was cleaved about 4-fold slower than the wild-type, whereas the

3 1454 Endoribonuclease of B. subtilis

1 2 3 4 5 FIG. 6. Reverse transcriptase analysis of HpaI RNA. Unlabeled

pJFD4 HpaI RNA was incubated with an aliquot from the phenyl- Sepharose fraction with either no M g + added (lane 1 ) or 3 mM Mg2‘ added (lanes 2 and 3). The cleaved RNAs were annealed to a labeled

Lane 2 contained 11.6 pmol of RNA substrate; lanes 1 and 3 contained oligonucleotide primer, which was extended with reverse transcriptase.

1.16 pmol of substrate. Lane 4, reverse transcriptase product synthe- sized on in vivo isolated RNA (50 pg). Lane 5, labeled primer alone. Arrow indicates the reverse transcriptase extension product.

substitutions that gave a purine at nt 215 (U + A or U + G) were cleaved a t a slightly slower rate (about 5- and 7-fold slower, respectively).

When the G residue at n t 216 was changed, however, a dramatic negative effect was found. Only the G 3 U mutation gave readily observable cleavage, which occurred a t a rate severalfold slower than pJFD4 XbaI RNA cleavage. The G - A and G --* C mutations showed barely detectable cleavage products, even after 30 min incubation a t 37 “C.

Heterologous Substrates-We tested the ability of the E. coli and B. subtilis RNase I11 enzymes to cleave the heterologous bacteriophage substrates. Uniformly-labeled T7 R1.l substrate was prepared from plasmid pAR1450 (see “Experimental Pro- cedures”). This was incubated with Bs-RNase I11 and E. coli RNase I11 under buffer conditions specific for each enzyme. Similarly, an equal amount of pJFD4 HpaI RNA was treated with both enzymes. The results (Fig. 7) showed that Bs-RNase I11 was able to cleave the T7 R1.l substrate. Based on the size of the 5”cleavage product, it appears that cleavage of T7 R1.l was at the same site by either enzyme. On the other hand, E. coli RNase I11 was unable to cleave pJFD4 HpaI RNA, despite the fact that, from the amounts of cleavage activity on the homologous substrates shown in Fig. 7, it is clear that the E. coli enzyme preparation contains a high level of cleavage activity. We could also not detect specific cleavage of pJFD4 HpaI RNA with E. coli RNase I11 when the reaction was done with the same NaCl concentration (100 mM) as the Bs-RNase I11 reaction (not shown).

DISCUSSION

We have undertaken a biochemical analysis of Bs-RNase 111, an endoribonuclease of B. subtilis that may be involved in the regulation of gene expression. The enzyme was purified to the extent that a single band was visible on a silver-stained gel. From the size of protein standards run in parallel on the gel, we estimate the molecular weight of Bs-RNase I11 to be about 29,000. This is similar to the previously reported size estimate of 27,000 (9). We cannot exclude the possibility that other B. subtilis proteins, and even endoribonucleases, are present in minute amounts in our most highly purified fraction. However, under standard assay conditions with many different RNA substrates, we have observed only single cleavage at the mapped site, suggesting that no other endonuclease is active. Efforts to clone the Bs-RNase I11 gene and overexpress its product are under way.

The primary cleavage site that was mapped by reverse transcriptase analysis on in vitro processed RNAs (between nucleotides 215 and 216) was precisely the same site as was mapped for the in vivo cleavage (11) (cf. Fig. 6, lanes 2 4 ) . This is different from the cleavage site reported by Panganiban and Whiteley (8), which was between nucleotides 199 and 200 in the numbering system shown in Fig. 2B. Although the methodology that we used to determine the cleavage site (reverse transcriptase mapping) differed substantially from that used ear- lier (5”end-labeling and RNA sequencing), we cannot ad- equately explain how such diverse results were obtained. Furthermore, based on their finding that Bs-RNase I11 cleaved in the upper loop portion, Panganiban and Whiteley explained thereby how Bs-RNase I11 and E. coli RNase I11 could recognize only their homologous substrates, since the E. coli RNase I11 cleavage was at a different site, in the internal loop segment. However, the mapped cleavage site that we show here for SP82 RNAs is, in fact, quite similar to the E. coli RNase I11 cleavage site on several T7 RNAs, which occurs in the internal bulge between a U and a G residue. Based on these data, we exam- ined the ability of Bs-RNase I11 to cleave T7 R1.l RNA. Bs- RNase I11 cleaved T7 R1.l RNA at the same site as E. coli RNase I11 (Fig. 7).

Most interestingly, the converse was not true. E. coli RNase I11 could not cleave the SP82 A site (Fig. 7). This represents a clear difference between the cleavage activity of these two enzymes and suggests that a comparative study of the specificity of the two RNase I11 enzymes could yield new information on the RNA-protein interactions that are necessary for binding and/or cleavage.

Deletion mutations in which nucleotides proximal to the cleavage site were retained but the potential for secondary structure was drastically reduced (e.g. upstream deletion pKH5 and Bal-31 deletion A223) showed no specific cleavage activity. More telling was the mutation of nt 219 from G to U in plasmid pJFD15, which is predicted to alter somewhat the secondary structure of the A site substrate. This change had a profound effect on the rate of cleavage (10-fold slower rate). This result suggests that there is a strong specificity for the double- stranded structure that is recognized by Bs-RNase I11 for binding and/or cleavage.

We found that the Bs-RNase I11 sites in the SP82 B and C regions were cleaved with only a 4-fold reduced rate (Fig. 5 ) . Although the B and C regions form structures whose overall appearance is predicted to be similar to that of the A region, there are many local differences which one might have thought would affect the rate of cleavage a t least as much as the G to U change in pJFD15 (which caused a 10-fold decrease in cleavage rare). We do not know enough about the “rules” for double- stranded RNA cleavage, and the energy-minimized structures

Endoribonuclease of B. subtilis 31455

TARI.E I1 Effect of mutations on Rs-RNase III cleavage

Construct name Type of mutation Decrease in cleavage rate

./i>/d

EG200 Deletion of sequences to 24 n t up- 1.5-2

P W 5 stream of stem loop structure

A223 A228 A234 pJFD15

Deletion of sequences to ScaI site No cleavage Bal-31 deletion from HpaI site No cleavage Bal-31 deletion from HpaI site 20 Bal-31 deletion from HpaI site 5 Point mutation in lower stem (nt 219) 10

T . ~ . E I11 Mutations at the L3s-RNa.w III cleavage site

Nucleotide change at cleavage site Decrease in cleavage rate

-fold 4 5 7

3-7 No cleavage No cleavage

pJFD4 Hpal RNA- 5"

"pAR1450 RNA

- 5'

pJFD4 HpaI RNA (left) and T7 R1.l RNA from pAR1450 (right) were Frc. 7. Cleavage of heterologous substrates. Equal amounts of

incubated with E. coli RNase I11 (ECR) or Bs-RNase I11 (BSR ). Aliquots were removed at the times indicated and run on a 5% denaturing polyacrylamide gel. Lanes marked (-) had no enzyme added. The pJFD4 HpaI RNA substrate was 278 nt long, with a 5"cleavage product (labeled 5 ') of 239 nt. The pAR1450 RNA substrate was 131 nt, with a 5'-cleavage product of 102 nt. In this exposure, the 3"cleavage products (39 nt for pJFD4 HpaI RNA and 29 nt for T7 R1.1) are barely visible near the bottom of the gel.

determined by the computer programs are not reliable enough, to be able to predict whether an RNA is a good substrate. It is noteworthy that the sequence immediately downstream of the cleavage site in all three SP82 regions is GGAGCC, whereas this sequence has been changed to GGAUCC in the case of pJFD15. The poor cleavage of pJDF15 RNA could imply that Bs-RNase I11 has some sequence specificity, which would also explain the dramatic effect on cleavage activity caused by changing the G residue at the cleavage site (Table 111). How- ever, the fact that Bs-RNase I11 can cleave T7 R1.l RNA, which does not contain this sequence, argues against such specificity. Other conserved sequences are also present in all three SP82 sites, the longest of these being GUUGCUAGUA(nt 182-191 in the A sequence) and GGGUGG (nt 203-208). Further mutagenesis experiments will be required to determine if these conserved sequences have any significance for Bs-RNase I11 cleavage. In this respect, we cannot exclude the possibility that the loss of cleavage activity for the pKH5 deletion (which is missing

conserved nt 182-191) is due to deletion of specific sequences rather than a change in secondary structure, as we have suggested.

The results with changes of the G residue at n t 216 were of great interest when compared with the effect of analogous changes at the site that is recognized by E. coli RNase I11 on the T7 R1.l substrate (5). As mentioned above, E. coli RNase I11 cleaves T7 R1.l RNA between a U and a G residue in a similarly positioned internal loop as that shown here for the SP82 substrate. Nicholson's group (5) found that a G --f A change caused only a 3.8-fold decrease in the rate of cleavage. We found that a G -> A change had a much more drastic effect, with barely detectable cleavage occurring after a 30 minute incubation. Furthermore, Nicholson's group found that changing of the G residue to either pyrimidine base (U or C) had no effect on the cleavage rate. We found that the G -> C change caused a virtually complete loss of activity, and the G -> U change caused a severalfold decrease in rate (the exact decrease varied among different experiments, but was between 3- and 7-fold). The fact that neither adenosine nor cytosine could substitute for guanosine at the cleavage site, but that substitution with uracil still gave some cleavage, might be explained by the presence of the hydroxyl group a t C-6 of guanosine and at C-4 of uracil. This group could be involved in the cleavage reaction. In any event, the differences between the effects of these changes in the E. coli and B. subtilis systems suggests that the cleavage mechanism may differ, despite the similarity in the overall structure and site of cleavage. Substitutions for the U residue at n t 215 with the other three nucleotides had less dramatic effects on the cleavage rate, indicating a less stringent require- ment for the 5' nucleotide at the cleavage site. The effect of changes at the analogous site in the E. coli system have not been reported. A complete analysis of the activity of the E. coli and B. subtilis RNase I11 enzymes on naturally occurring substrates from E. coli and B. subtilis will likely be highly informative.

Acknowledgments-Plasmids of the pKH series and the Bal-31 deletion mutations were made by Kim Hue, whose expert technical assistance is gratefully acknowledged. We appreciate greatly Dr. R. Kohanski's invaluable advice in the protein purification procedure. We thank Dr. A. Sloma for the protease-deficient 23. suhtilis strain and Dr. A. Nicholson for purified E. roli RNase I11 and plasmid pAR1450.

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