366
PROCESSING OF RIBOSOMAL R N A s
[30]
nucleotides at each end. Additional species found in cells in which protein synthesis is inhibited have 1, 2, or 3 extra 5'-nucleotides. 21 No assay is reported for the final maturation (which may conceivably require ribosomes or polysomes as a substrate). 21 j. Feunteun, B. R. Jordan, and R. J. Monier, J. Mol. Biol. 70, 465 (1972).
[30] R i b o s o m a l R N A T e r m i n a l M a t u r a s e : R i b o n u c l e a s e M 5 from Bacillus subtilis B y N O R M A N R . PACE a n d BERNADETTE PACE
The ribosomal RNAs (rRNAs) of all cells are acted upon by several RNA processing nucleases and nucleotide-modifying enzymes during the formation of the mature ribosome. 1 Although some of these enzymes (e.g., RNase III) are capable of using naked RNA as a substrate, others require ribonucleoprotein (RNP) substrates. The terminal maturases, the nucleases that produce the mature termini of the rRNAs by removing precursor-specific RNA sequences, are examples of the latter. The general requirement of the terminal maturases for RNP substrates is best evidenced by the fact that the immediate precursors of the mature rRNAs accumulate in cells treated with inhibitors of protein synthesis. The proteins required for the formation of productive substrates for the terminal rRNA maturases probably are generally ribosomal proteins, known to associate with the nascent rRNAs during transcription. The terminal rRNA maturases have received relatively little study, in large part because of the technical hurdle of isolating or reconstructing precursor rRNA-containing RNPs for their assay. Enzymatic activities that result in the terminal maturation of 16 and 23 S rRNA in Escherichia coli have been demonstrated in vitro using partially purified extracts; 2-5 however, little information is available regarding the properties of the enzymes. The only rRNA maturase that so far has been characterized in any detail is RNase M5 from Bacillus subtilis. 6 I T. C. King, R. Sirdeskmukh, and D. Schlessinger, Microbiol. Rev. 50, 428 (1986). 2 B. Meyhack, I. Meyhack, and D. Apirion, FEBS Lett. 49, 215 (1974). 3 F. Hayes and M. Vasseur, Eur. J. Biochem. 61, 433 (1976). 4 A. E. Dahlberg, J. E. Dahlberg, E. Lund, H. Tokimatsu, A. B. Rabson, P. C. Calvert, F. Reynolds, and M. Zahalak, Proc. Natl. Acad. Sci. U.S.A. 75, 3598 (1978). 5 R. Sirdeskmukh and D. Schlessinger, Nucleic Acids Res. 13, 5041 (1985). 6 M. L. Sogin, B. Pace, and N. R. Pace, J. Biol. Chem. 252, 1350 (1977).
METHODS IN ENZYMOLOGY, VOL. 181
Copyright © 1990by Academic Press, Inc. All rights of reproduction in any form reserved.
[30]
B. subtilis R N a s e M5
367
RNase M5 precisely removes both 5'- and 3'-terminal precursor-specific sequences from 5 S rRNA (Fig. 1). 7 This B. subtilis enzyme was chosen for study as a model rRNA maturase because of the relatively small size, hence manipulable nature, of its substrates. Three immediate precursors of 5 S rRNA, about 150, 180, and 240 nucleotides in length, accumulate in B. subtilis in the presence of inhibitors of protein synthesis. 8 The variation in size of the precursors occurs because they are derived from different 5 S rRNA genes with somewhat different precursor sequences. Isotopically labeled pre-5 S rRNA added to extracts of B. subtilis undergoes precise maturation, so the required components could be isolated. 6 Two protein components proved to be required for 5 S rRNA maturation. One of these, originally termed or, is the actual endonuclease RNase M 5 . 9 The second, termed fl in early reports, is ribosomal protein BL16.10 This ribosomal protein is the homolog of the well-studied E. coli 5 S rRNA-binding ribosomal protein EL18,11 which, in fact, can substitute for BL16 in the RNase M5 reaction. The ribosomal proteins bind to the pre-5 S rRNA to form the RNP substrate for the enzyme. The other two known 5 S rRNA-binding proteins from E. coli, EL25 and EL5, alone or in combination with EL18 (or BL16) do not potentiate cleavage of the pre-5 S rRNA by RNase M5. Protein EL5 inhibits the RNase M5 reaction, probably because it binds to the helical segment of the RNA at which RNase M5 acts (Fig. 1). 9 The requirement by RNase M5 for the RNP substrate in vitro is nearly absolute under physiological conditions. However, in the presence of 2030% dimethyl sulfoxide the maturase accurately cleaves pre-5 S rRNA in the absence of BL16. 9 Thus, the major structural features of the substrate that are recognized by the maturase reside in the RNA. The entirety of the pre-5 S rRNA is not required for recognition and action by RNase M5, however. Substantial segments of the substrate RNA can be deleted without abolishing susceptibility to the enzyme. 7,j2 None of the precursorspecific nucleotides is specifically required for action, so long as the double-helical character of the substrate sites is maintained.~3 7 B. Meyhack, B. Pace, and N. R. Pace, Biochemistry 16, 5009 (1977). 8 N. R. Pace, M. C. Pato, M. McKibbin, and C. W. Radcliffe, J. Mol. Biol. 75, 619 (1973). 9 B. Pace, D. A. Stahl, and N. R. Pace, J. Biol. Chem. 259, 11454 (1984). 10 D. A. Stahl, B. Pace, T. Marsh, and N. R. Pace, J. Biol. Chem. 259, 11448 (1984). H R. A. Zimmerman, this series, Vol. 59, p. 551. ~2 B. Meyhack and N. R. Pace, Biochemistry 17, 5804 (1978). 13 D. A. Stahl, B. Meyhack, and N. R. Pace, Proc. Natl. Acad. Sci. U.S.A. 77, 5644 (1980).
%
,% Z r~
% " , % = .., o
"-' ..
0.g ,_,
__
,--,
,
,...,
u~
.,~ ¢~.~:~
%,
\ ~o~..~ o
~,o =~.~
E
~-u
o
~-o
E ¢~¢,j
0 L
I
b-, Z~
,u /
\
~
,:," '%"
(/I
~
"J
z
/
-J
g
\
o2~
-1
Z ~ I I I I I l
0"% ~Z,"% . . d (,~
.%
e,
::th
o
[30]
B. subtilis RNase M5
369
Substrates and Assays for Terminal Maturases The simplest assay for terminal processing nucleases is achieved by incubating an isotopically labeled precursor rRNA or RNP with appropriate cell extracts and scoring the production of mature-size products by polyacrylamide gel electrophoresis. Early work with RNase M5 utilized as a substrate uniformly 32p-labeled pre-5 S rRNAs that accumulate in B. subtilis in the presence of chloramphenicol (an inhibitor of protein synthesis). The purification of pre-5 S rRNAs by phenol extraction and polyacrylamide gel electrophoresis as well as the gel assay for RNase M5 have been detailed previously. 6 Polyacrylamide gel assays are unwieldy for large numbers of samples, however. When it was discovered that the precursor-specific segments had no role in the recognition of pre-5 S rRNA by RNase M5, lz it was possible to fabricate a more convenient assay substrate (Fig. 2). As detailed, 14 RNA ligase was used to add a synthetic oligonucleotide, usually U U U G (the in vivo sequence), to the 5' end of mature 5 S rRNA. The new 5' end then was labeled using [3,_ 32p]ATP and polynucleotide kinase, and release of the synthetic "precursor" segment by RNase M5 was scored by loss of acid-insoluble radioactivity or by analysis using polyethyleneimine thin-layer chromatography. In the latter assay, with the chromatography solvents employed, the uncleaved substrate (120 nucleotides) remains at the origin of the chromatogram, and the released, isotopically labeled oligonucleotide (4 nucleotides) migrates at a characteristic rate (Fig. 2, inset). Quantitative results are obtained by scissoring out or scraping radioactive spots that are located by autoradiography and counting them in a scintillation spectrometer. The enzymatic activities that produce the mature termini of 16 S rRNA in E. coli have been assayed in vitro using as substrates pulselabeled preribosome particles 2,3 or ribosomes derived from a mutant defective in the maturation of the 5' end of 16 S rRNA. 4 Naked pre-16 S rRNA is not a substrate for these maturation reactions. There have been no reports of attempts to reconstitute substrates for maturases using purified ribosomal proteins and pre-rRNAs isolated from cells or synthesized in vitro with transcription vectors; however, this approach seems viable. The pre-rRNA-containing RNPs that accumulate in cells in the absence of protein synthesis are not likely to be effective substrates, since they probably do not contain a normal complement of ribosomal proteins. 14 B. Meyhack, B. Pace, O. C. Uhlenbeck, and N. R. Pace, Proc. Natl. Acad. Sci. U.S.A. 75, 3045 (1978).
370
PROCESSING OF RIBOSOMALRNAs
[30]
.C-A-UA -¢
C
5' 10' 15'
4-- pU3GoH
c-Ac-" V
~- Origin
RNa se M5
C
,c',:G.A u
~G.¢_G.A 'A
KU-A-G~- °,G-~,
J-U-G-~U-U-U-G-G-U-G-G-C-G
_,G'C
HO
~,°o~ UoU
U4J' FIG. 2. Assay of RNase M5 using a partially synthetic substrate. As described in the text, a synthetic oligonucleotide (UUUG in the example shown) is appended to the 5' end of mature 5 S rRNA using RNA ligase. Then the artificial substrate is labeled at its 5' end using [~/-32p]ATP and polynucleotide kinase. Incubation with RNase M5 releases the synthetic precursor segment, which migrates at a characteristic rate during thin-layer chromatography on polyethyleneimine-impregnated cellulose (inset).
Nonionic Detergents and Rare Proteins T h e t e r m i n a l r R N A m a t u r a s e s a r e r a r e : e a c h B. subtilis cell p r o b a b l y c o n t a i n s no m o r e t h a n a b o u t I00 m o l e c u l e s o f R N a s e M5. T h e e x t e n s i v e p u r i f i c a t i o n o f r a r e e n z y m e s s u c h as R N a s e M5 i n e v i t a b l y r e s u l t s in l o w s o l u t i o n c o n c e n t r a t i o n s o f p r o t e i n , a c o n d i t i o n t h a t is c o n s i d e r e d to p r e -
[30]
B. subtilis RNase M5
371
dispose enzymes to denaturation and loss of activity. (Hence, bovine serum albumin commonly is added to pure enzymes.) During early studies RNase M5, too, seemed "unstable" at low concentrations of protein. 6 However, this proved to be due to the tenacious adherence of the enzyme to glass and plastic surfaces. Losses in enzyme activity during purification and storage could be countered by the inclusion of a nonionic detergent in all buffers that are used for dilute solutions of proteins. 9 Two detergents, Nonidet P-40 (NP-40) and Brij 58, at concentrations of 0.01-0.1% were interchangeably employed in studies with RNase M5. NP-40 has satisfactory solubility properties in all buffers utilized, but it absorbs light strongly in the ultraviolet. Brij 58 has no significant UV absorbance, but it forms micelles at high ionic strengths and in phosphate-containing buffers in the cold. The inclusion of a detergent in buffers is to be recommended generally for the purification of proteins or other macromolecules. We believe that many anecdotes regarding enzyme denaturation at low solution concentrations of protein can be accounted for by adsorption to surfaces: storage tube walls, flow tubes, chromatographic supports, etc. Experimental Procedures Assay o f R N a s e M5. Ten-microliter reactions contain 10 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HC1) (pH 7.4), 5 mM MgC12, 30% (w/v) glycerol, 2 mM dithiothreitol, 0.05% Brij 58, 1-10 ng 32p-labeled pre-5 S rRNA (at a specific radioactivity o f - 1 0 6 cpm//.~g RNA), and test amounts of RNase M5 and BL16 proteins. Reactions are mixed on ice, then incubated for appropriate times at 37°. In the thin-layer chromatographic assay, ~3 reactions are terminated by chilling in an ice bath and adding I/zl of 0.5% formic acid, and aliquots of the reactions are spotted onto plastic-backed, polyethyleneimine-impregnated thin-layer plates (Brinkman No. 801063). Glycerol and salts are washed off the origin by ascending chromatography with water to a few centimeters past the origin, then, without drying, ascending chromatography is continued with 1 M formic acid adjusted to pH 4.3 with pyridine. Reaction products are visualized by autoradiography. In the use of the polyacrylamide gel electrophoresis assay, reactions are terminated by adding 1 /~l of 10% sodium dodecyl sulfate (SDS) 50% (w/v) sucrose, and products are resolved by electrophoresis in 8% polyacrylamide gels as detailed previously. 6 Protein Gel Electrophoresis. Protein purifications are monitored by silver staining 15 on 15% polyacrylamide gels following electrophoresis in the second dimension buffer of O'Farrell.16 The B. subtilis fl protein was 15 B. R. Oakley, D. R. Kirsch, and N. R. Morris, Anal. Biochem. 105, 361 (1980). 16 p. H. O'Farrell, J. Biol. Chem. 250, 4007 (1975).
372
PROCESSING OF RIBOSOMAL R N A s
[30]
identified as BL16 ribosomal protein by Dr. Eiko Otaka (Research Institute for Nuclear Medicine and Biology, Hiroshima University, Japan), using the two-dimensional polyacrylamide gel electrophoresis system of Kaltschmidt and Wittmann. 17 Purification ofRNase M5. Frozen B. subtilis 168 cell paste (175 g) is thawed in 170 ml of PA Buffer [50 mM Tris-HCl (pH 7.4), 20 mM MgCI2, 5% (w/v) glycerol, 60 mM NH4CI] adjusted to 200 mM NHnC1 and 1/xg/ml DNase I (Sigma Type II). All enzyme solutions are manipulated and stored at 4 ° or lower. Cells are broken by passage through an Aminco French pressure cell at 20,000 psi. Cellular debris and ribosomes are removed by centrifugation, first at 18,000 rpm in a Sorvall SS34 rotor for 40 rain and then at 45,000 rpm for 6 hr in a Beckman 50.2 Ti rotor. The supernatant is dialyzed overnight against 2 changes of PA buffer. After a fine precipitate is removed by centrifugation at 10,000 rpm for 20 min, the dialyzate is loaded onto a 500-ml DEAE-cellulose column (Whatman DE-52) equilibrated in PA buffer, then washed with the same buffer. Flow-through and wash fractions are assayed for RNase M5 activity (supplementing assay reactions with ribosomal protein BL16), and the relevant fractions are pooled. A nonionic detergent, NP-40 (Sigma, St. Louis, MO) or Brij 58 (Sigma), is then added to 0.1%, and is included in all subsequent buffers in order to prevent adsorption to surfaces at low concentrations of protein. After dialysis against SB buffer [50 mM Tris-HC1 (pH 8.0), 30 mM NHaCI, 4 mM 2-mercaptoethanol, 0.1 mM ethylenediaminetetraaceticacid (EDTA), 0. I% NP-40], the enzyme preparation is loaded onto a 150ml phosphocellulose column (Whatman P-11). SB buffer is used for column equilibration, for washing after loading, and for the linear 30-600 mM NH4CI gradient. Column fractions are assayed, and the RNase M5 peak, at about 300 mM NHaC1, is pooled and dialyzed against SB buffer. It is then loaded onto a 5-ml Affi-Gel Blue (Bio-Rad, Richmond, CA), column, which has been washed extensively with SB buffer containing 600 mM NHaCI in order to strip UV-absorbing materials from the matrix and then equilibrated to SB buffer before use. The peak RNase M5 activity elutes at 150 mM in a 30-300 mM NH4CI gradient in SB buffer. Most of the reported studies of RNase M5 used enzyme at this level of purity. Preparations are greatly enriched in RNase M5 activity and free of other nucleases. A spectrophotometric scan of a silver-stained, polyacrylamide gel electrophoresis track of such an RNase M5 preparation indicated that the enzyme constitutes about 3% of the silver-staining material, presuming equivalent staining of all bands present. This type of estimate is used to calculate nominal enzyme concentrations in RNase M5 17 E. Kaltschmidt and H. G. Wittmann, Anal. Biochem. 36, 401 (1970).
[30]
B. subtilis RNase M5
373
reactions. It is not known what fraction of the protein in the RNase M5 band is in fact catalytically active. Further purification can be achieved by sequential chromatography, at pH 6.5 and 4.0, on 2-ml phosphocellulose columns, using 0-300 mM NaCI gradients in 50 mM sodium phosphate buffer, 4 mM 2-mercaptoethanol, 0. I% NP-40, 6 M urea. A 1-ml carboxymethylcellulose (Whatman CM-52) column with a 0-300 mM NaC1 gradient in 50 mM sodium acetate (pH 4.0), 4 mM 2-mercaptoethanol, 0.1% NP-40, 6 M urea also can afford some purification. Final purification of the RNase M5 activity to homogeneity is achieved by denaturing (SDS) polyacrylamide gel electrophoresis, recovering the protein according to the protocol of Hager and Burgess.~8 A single gel band of Mr 24,000 is highly active against the BL16--pre-rRNA RNP substrate; however, the number of peptide chains required to form the active enzyme is not known. Purification of Ribosomal Proteins BL16 and EL18. Ribosomal proteins BL16 and EL18 originally were purified as activities (/3) that are required for the RNase M5 reaction. When it was discovered that the/3 activities are ribosomal proteins, the following protocol was devised. It is similar to methods commonly used to purify ribosomal proteins.11 Frozen E. coil or B. subtilis cell paste (50 g) is thawed in 50 ml of buffer B [20 mM Tris-HC1 (pH 7.8), 30 mM NH4CI, 10 mM magnesium acetate, 10 mM 2-mercaptoethanol] containing 1 /xg/ml DNase I (Sigma Type II) and broken by passage through an Aminco French pressure cell at 20,000 psi. All steps in the purification schedule are carried out at or below 4 °. The lysate is diluted with 50 ml of buffer B and centrifuged for 20 min at 18,000 rpm in a Sorvall SS34 rotor. The supernatant is centrifuged for 5 hr at 45,000 rpm in a Beckman 50.2 Ti rotor, and the ribosome pellet resuspended in buffer B. The NH4CI concentration is adjusted to 1 M and the volume to 120 ml by the addition of buffer B, and the ribosomes are again pelleted. The salt-washed ribosomes are resuspended in 75 ml of buffer B. After dialysis overnight against 20 mM Tris-HC1 (pH 7.8), 10 mM NH4CI, 2 mM magnesium acetate, 10 mM 2-mercaptoethanol, 0.05% Brij 58 (or NP-40), ribosomes are dispersed by addition of urea crystals to 8.5 M. After 24 hr on ice, the urea concentration is reduced to 6.5 M by addition of 50 mM sodium phosphate (pH 6.5), and a fine precipitate is removed by centrifugation for 5 min at 18,000 rpm in a Sorvall SS34 rotor. Approximately 25,000 A260units of supernatant is loaded onto a 500-ml phosphocellulose column equilibrated with 50 mM sodium phosphate (pH 6.5), 6 M urea, 2 mM methylamine, 0.05% NP-40 and washed with several hundred milliliters of the same buffer. Proteins are eluted with a 0-700 is D. A. Hager and R. R. Burgess, Anal. Biochem. 109, 76 (1980).
374
PROCESSINGOF RIBOSOMALRNAs
[30]
mM NH4CI gradient in the same buffer; the activities that complement the RNase M5 reaction elute at about 300 mM. Peak activity-containing fractions are pooled, dialyzed against 50 mM acetic acid (pH 3), 0.1 mM EDTA, 0.1% NP-40, 30 mM NH4C1, loaded onto a 15-ml phosphocellulose column in the same buffer, and eluted with a 0.03-1.2 M gradient of NH4CI in that buffer. Both BL16 and EL18 elute late, at approximately 800 mM NH4CI, and are nearly pure at this stage. A third column chromatography is carried out on a 2-ml phosphocellulose column in 40 mM TrisHC1 (pH 7.4), 0.1 mM EDTA, 0.1% NP-40, 30 mM NH4CI, eluting with a 30-700 mM NH4CI gradient; the ribosomal proteins elute at approximately 400 mM. A fourth column, required only for rigorous purification of the ELI8, is the same as the third except that all buffers contain 6 M urea. The EL18 elutes at approximately 150 mM in the 20-400 mM NH4CI gradient. Pooled fractions are dialyzed against buffer B containing 0.1% NP-40 and stored as aliquots frozen at -70 °. Acknowledgments The authors' research is supported by National Institutes of Health Grant GM34527to N.R.P. We thank David Smithfor artwork.
PROCESSING OF RIBOSOMAL R N A s
[30]
nucleotides at each end. Additional species found in cells in which protein synthesis is inhibited have 1, 2, or 3 extra 5'-nucleotides. 21 No assay is reported for the final maturation (which may conceivably require ribosomes or polysomes as a substrate). 21 j. Feunteun, B. R. Jordan, and R. J. Monier, J. Mol. Biol. 70, 465 (1972).
[30] R i b o s o m a l R N A T e r m i n a l M a t u r a s e : R i b o n u c l e a s e M 5 from Bacillus subtilis B y N O R M A N R . PACE a n d BERNADETTE PACE
The ribosomal RNAs (rRNAs) of all cells are acted upon by several RNA processing nucleases and nucleotide-modifying enzymes during the formation of the mature ribosome. 1 Although some of these enzymes (e.g., RNase III) are capable of using naked RNA as a substrate, others require ribonucleoprotein (RNP) substrates. The terminal maturases, the nucleases that produce the mature termini of the rRNAs by removing precursor-specific RNA sequences, are examples of the latter. The general requirement of the terminal maturases for RNP substrates is best evidenced by the fact that the immediate precursors of the mature rRNAs accumulate in cells treated with inhibitors of protein synthesis. The proteins required for the formation of productive substrates for the terminal rRNA maturases probably are generally ribosomal proteins, known to associate with the nascent rRNAs during transcription. The terminal rRNA maturases have received relatively little study, in large part because of the technical hurdle of isolating or reconstructing precursor rRNA-containing RNPs for their assay. Enzymatic activities that result in the terminal maturation of 16 and 23 S rRNA in Escherichia coli have been demonstrated in vitro using partially purified extracts; 2-5 however, little information is available regarding the properties of the enzymes. The only rRNA maturase that so far has been characterized in any detail is RNase M5 from Bacillus subtilis. 6 I T. C. King, R. Sirdeskmukh, and D. Schlessinger, Microbiol. Rev. 50, 428 (1986). 2 B. Meyhack, I. Meyhack, and D. Apirion, FEBS Lett. 49, 215 (1974). 3 F. Hayes and M. Vasseur, Eur. J. Biochem. 61, 433 (1976). 4 A. E. Dahlberg, J. E. Dahlberg, E. Lund, H. Tokimatsu, A. B. Rabson, P. C. Calvert, F. Reynolds, and M. Zahalak, Proc. Natl. Acad. Sci. U.S.A. 75, 3598 (1978). 5 R. Sirdeskmukh and D. Schlessinger, Nucleic Acids Res. 13, 5041 (1985). 6 M. L. Sogin, B. Pace, and N. R. Pace, J. Biol. Chem. 252, 1350 (1977).
METHODS IN ENZYMOLOGY, VOL. 181
Copyright © 1990by Academic Press, Inc. All rights of reproduction in any form reserved.
[30]
B. subtilis R N a s e M5
367
RNase M5 precisely removes both 5'- and 3'-terminal precursor-specific sequences from 5 S rRNA (Fig. 1). 7 This B. subtilis enzyme was chosen for study as a model rRNA maturase because of the relatively small size, hence manipulable nature, of its substrates. Three immediate precursors of 5 S rRNA, about 150, 180, and 240 nucleotides in length, accumulate in B. subtilis in the presence of inhibitors of protein synthesis. 8 The variation in size of the precursors occurs because they are derived from different 5 S rRNA genes with somewhat different precursor sequences. Isotopically labeled pre-5 S rRNA added to extracts of B. subtilis undergoes precise maturation, so the required components could be isolated. 6 Two protein components proved to be required for 5 S rRNA maturation. One of these, originally termed or, is the actual endonuclease RNase M 5 . 9 The second, termed fl in early reports, is ribosomal protein BL16.10 This ribosomal protein is the homolog of the well-studied E. coli 5 S rRNA-binding ribosomal protein EL18,11 which, in fact, can substitute for BL16 in the RNase M5 reaction. The ribosomal proteins bind to the pre-5 S rRNA to form the RNP substrate for the enzyme. The other two known 5 S rRNA-binding proteins from E. coli, EL25 and EL5, alone or in combination with EL18 (or BL16) do not potentiate cleavage of the pre-5 S rRNA by RNase M5. Protein EL5 inhibits the RNase M5 reaction, probably because it binds to the helical segment of the RNA at which RNase M5 acts (Fig. 1). 9 The requirement by RNase M5 for the RNP substrate in vitro is nearly absolute under physiological conditions. However, in the presence of 2030% dimethyl sulfoxide the maturase accurately cleaves pre-5 S rRNA in the absence of BL16. 9 Thus, the major structural features of the substrate that are recognized by the maturase reside in the RNA. The entirety of the pre-5 S rRNA is not required for recognition and action by RNase M5, however. Substantial segments of the substrate RNA can be deleted without abolishing susceptibility to the enzyme. 7,j2 None of the precursorspecific nucleotides is specifically required for action, so long as the double-helical character of the substrate sites is maintained.~3 7 B. Meyhack, B. Pace, and N. R. Pace, Biochemistry 16, 5009 (1977). 8 N. R. Pace, M. C. Pato, M. McKibbin, and C. W. Radcliffe, J. Mol. Biol. 75, 619 (1973). 9 B. Pace, D. A. Stahl, and N. R. Pace, J. Biol. Chem. 259, 11454 (1984). 10 D. A. Stahl, B. Pace, T. Marsh, and N. R. Pace, J. Biol. Chem. 259, 11448 (1984). H R. A. Zimmerman, this series, Vol. 59, p. 551. ~2 B. Meyhack and N. R. Pace, Biochemistry 17, 5804 (1978). 13 D. A. Stahl, B. Meyhack, and N. R. Pace, Proc. Natl. Acad. Sci. U.S.A. 77, 5644 (1980).
%
,% Z r~
% " , % = .., o
"-' ..
0.g ,_,
__
,--,
,
,...,
u~
.,~ ¢~.~:~
%,
\ ~o~..~ o
~,o =~.~
E
~-u
o
~-o
E ¢~¢,j
0 L
I
b-, Z~
,u /
\
~
,:," '%"
(/I
~
"J
z
/
-J
g
\
o2~
-1
Z ~ I I I I I l
0"% ~Z,"% . . d (,~
.%
e,
::th
o
[30]
B. subtilis RNase M5
369
Substrates and Assays for Terminal Maturases The simplest assay for terminal processing nucleases is achieved by incubating an isotopically labeled precursor rRNA or RNP with appropriate cell extracts and scoring the production of mature-size products by polyacrylamide gel electrophoresis. Early work with RNase M5 utilized as a substrate uniformly 32p-labeled pre-5 S rRNAs that accumulate in B. subtilis in the presence of chloramphenicol (an inhibitor of protein synthesis). The purification of pre-5 S rRNAs by phenol extraction and polyacrylamide gel electrophoresis as well as the gel assay for RNase M5 have been detailed previously. 6 Polyacrylamide gel assays are unwieldy for large numbers of samples, however. When it was discovered that the precursor-specific segments had no role in the recognition of pre-5 S rRNA by RNase M5, lz it was possible to fabricate a more convenient assay substrate (Fig. 2). As detailed, 14 RNA ligase was used to add a synthetic oligonucleotide, usually U U U G (the in vivo sequence), to the 5' end of mature 5 S rRNA. The new 5' end then was labeled using [3,_ 32p]ATP and polynucleotide kinase, and release of the synthetic "precursor" segment by RNase M5 was scored by loss of acid-insoluble radioactivity or by analysis using polyethyleneimine thin-layer chromatography. In the latter assay, with the chromatography solvents employed, the uncleaved substrate (120 nucleotides) remains at the origin of the chromatogram, and the released, isotopically labeled oligonucleotide (4 nucleotides) migrates at a characteristic rate (Fig. 2, inset). Quantitative results are obtained by scissoring out or scraping radioactive spots that are located by autoradiography and counting them in a scintillation spectrometer. The enzymatic activities that produce the mature termini of 16 S rRNA in E. coli have been assayed in vitro using as substrates pulselabeled preribosome particles 2,3 or ribosomes derived from a mutant defective in the maturation of the 5' end of 16 S rRNA. 4 Naked pre-16 S rRNA is not a substrate for these maturation reactions. There have been no reports of attempts to reconstitute substrates for maturases using purified ribosomal proteins and pre-rRNAs isolated from cells or synthesized in vitro with transcription vectors; however, this approach seems viable. The pre-rRNA-containing RNPs that accumulate in cells in the absence of protein synthesis are not likely to be effective substrates, since they probably do not contain a normal complement of ribosomal proteins. 14 B. Meyhack, B. Pace, O. C. Uhlenbeck, and N. R. Pace, Proc. Natl. Acad. Sci. U.S.A. 75, 3045 (1978).
370
PROCESSING OF RIBOSOMALRNAs
[30]
.C-A-UA -¢
C
5' 10' 15'
4-- pU3GoH
c-Ac-" V
~- Origin
RNa se M5
C
,c',:G.A u
~G.¢_G.A 'A
KU-A-G~- °,G-~,
J-U-G-~U-U-U-G-G-U-G-G-C-G
_,G'C
HO
~,°o~ UoU
U4J' FIG. 2. Assay of RNase M5 using a partially synthetic substrate. As described in the text, a synthetic oligonucleotide (UUUG in the example shown) is appended to the 5' end of mature 5 S rRNA using RNA ligase. Then the artificial substrate is labeled at its 5' end using [~/-32p]ATP and polynucleotide kinase. Incubation with RNase M5 releases the synthetic precursor segment, which migrates at a characteristic rate during thin-layer chromatography on polyethyleneimine-impregnated cellulose (inset).
Nonionic Detergents and Rare Proteins T h e t e r m i n a l r R N A m a t u r a s e s a r e r a r e : e a c h B. subtilis cell p r o b a b l y c o n t a i n s no m o r e t h a n a b o u t I00 m o l e c u l e s o f R N a s e M5. T h e e x t e n s i v e p u r i f i c a t i o n o f r a r e e n z y m e s s u c h as R N a s e M5 i n e v i t a b l y r e s u l t s in l o w s o l u t i o n c o n c e n t r a t i o n s o f p r o t e i n , a c o n d i t i o n t h a t is c o n s i d e r e d to p r e -
[30]
B. subtilis RNase M5
371
dispose enzymes to denaturation and loss of activity. (Hence, bovine serum albumin commonly is added to pure enzymes.) During early studies RNase M5, too, seemed "unstable" at low concentrations of protein. 6 However, this proved to be due to the tenacious adherence of the enzyme to glass and plastic surfaces. Losses in enzyme activity during purification and storage could be countered by the inclusion of a nonionic detergent in all buffers that are used for dilute solutions of proteins. 9 Two detergents, Nonidet P-40 (NP-40) and Brij 58, at concentrations of 0.01-0.1% were interchangeably employed in studies with RNase M5. NP-40 has satisfactory solubility properties in all buffers utilized, but it absorbs light strongly in the ultraviolet. Brij 58 has no significant UV absorbance, but it forms micelles at high ionic strengths and in phosphate-containing buffers in the cold. The inclusion of a detergent in buffers is to be recommended generally for the purification of proteins or other macromolecules. We believe that many anecdotes regarding enzyme denaturation at low solution concentrations of protein can be accounted for by adsorption to surfaces: storage tube walls, flow tubes, chromatographic supports, etc. Experimental Procedures Assay o f R N a s e M5. Ten-microliter reactions contain 10 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HC1) (pH 7.4), 5 mM MgC12, 30% (w/v) glycerol, 2 mM dithiothreitol, 0.05% Brij 58, 1-10 ng 32p-labeled pre-5 S rRNA (at a specific radioactivity o f - 1 0 6 cpm//.~g RNA), and test amounts of RNase M5 and BL16 proteins. Reactions are mixed on ice, then incubated for appropriate times at 37°. In the thin-layer chromatographic assay, ~3 reactions are terminated by chilling in an ice bath and adding I/zl of 0.5% formic acid, and aliquots of the reactions are spotted onto plastic-backed, polyethyleneimine-impregnated thin-layer plates (Brinkman No. 801063). Glycerol and salts are washed off the origin by ascending chromatography with water to a few centimeters past the origin, then, without drying, ascending chromatography is continued with 1 M formic acid adjusted to pH 4.3 with pyridine. Reaction products are visualized by autoradiography. In the use of the polyacrylamide gel electrophoresis assay, reactions are terminated by adding 1 /~l of 10% sodium dodecyl sulfate (SDS) 50% (w/v) sucrose, and products are resolved by electrophoresis in 8% polyacrylamide gels as detailed previously. 6 Protein Gel Electrophoresis. Protein purifications are monitored by silver staining 15 on 15% polyacrylamide gels following electrophoresis in the second dimension buffer of O'Farrell.16 The B. subtilis fl protein was 15 B. R. Oakley, D. R. Kirsch, and N. R. Morris, Anal. Biochem. 105, 361 (1980). 16 p. H. O'Farrell, J. Biol. Chem. 250, 4007 (1975).
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PROCESSING OF RIBOSOMAL R N A s
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identified as BL16 ribosomal protein by Dr. Eiko Otaka (Research Institute for Nuclear Medicine and Biology, Hiroshima University, Japan), using the two-dimensional polyacrylamide gel electrophoresis system of Kaltschmidt and Wittmann. 17 Purification ofRNase M5. Frozen B. subtilis 168 cell paste (175 g) is thawed in 170 ml of PA Buffer [50 mM Tris-HCl (pH 7.4), 20 mM MgCI2, 5% (w/v) glycerol, 60 mM NH4CI] adjusted to 200 mM NHnC1 and 1/xg/ml DNase I (Sigma Type II). All enzyme solutions are manipulated and stored at 4 ° or lower. Cells are broken by passage through an Aminco French pressure cell at 20,000 psi. Cellular debris and ribosomes are removed by centrifugation, first at 18,000 rpm in a Sorvall SS34 rotor for 40 rain and then at 45,000 rpm for 6 hr in a Beckman 50.2 Ti rotor. The supernatant is dialyzed overnight against 2 changes of PA buffer. After a fine precipitate is removed by centrifugation at 10,000 rpm for 20 min, the dialyzate is loaded onto a 500-ml DEAE-cellulose column (Whatman DE-52) equilibrated in PA buffer, then washed with the same buffer. Flow-through and wash fractions are assayed for RNase M5 activity (supplementing assay reactions with ribosomal protein BL16), and the relevant fractions are pooled. A nonionic detergent, NP-40 (Sigma, St. Louis, MO) or Brij 58 (Sigma), is then added to 0.1%, and is included in all subsequent buffers in order to prevent adsorption to surfaces at low concentrations of protein. After dialysis against SB buffer [50 mM Tris-HC1 (pH 8.0), 30 mM NHaCI, 4 mM 2-mercaptoethanol, 0.1 mM ethylenediaminetetraaceticacid (EDTA), 0. I% NP-40], the enzyme preparation is loaded onto a 150ml phosphocellulose column (Whatman P-11). SB buffer is used for column equilibration, for washing after loading, and for the linear 30-600 mM NH4CI gradient. Column fractions are assayed, and the RNase M5 peak, at about 300 mM NHaC1, is pooled and dialyzed against SB buffer. It is then loaded onto a 5-ml Affi-Gel Blue (Bio-Rad, Richmond, CA), column, which has been washed extensively with SB buffer containing 600 mM NHaCI in order to strip UV-absorbing materials from the matrix and then equilibrated to SB buffer before use. The peak RNase M5 activity elutes at 150 mM in a 30-300 mM NH4CI gradient in SB buffer. Most of the reported studies of RNase M5 used enzyme at this level of purity. Preparations are greatly enriched in RNase M5 activity and free of other nucleases. A spectrophotometric scan of a silver-stained, polyacrylamide gel electrophoresis track of such an RNase M5 preparation indicated that the enzyme constitutes about 3% of the silver-staining material, presuming equivalent staining of all bands present. This type of estimate is used to calculate nominal enzyme concentrations in RNase M5 17 E. Kaltschmidt and H. G. Wittmann, Anal. Biochem. 36, 401 (1970).
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B. subtilis RNase M5
373
reactions. It is not known what fraction of the protein in the RNase M5 band is in fact catalytically active. Further purification can be achieved by sequential chromatography, at pH 6.5 and 4.0, on 2-ml phosphocellulose columns, using 0-300 mM NaCI gradients in 50 mM sodium phosphate buffer, 4 mM 2-mercaptoethanol, 0. I% NP-40, 6 M urea. A 1-ml carboxymethylcellulose (Whatman CM-52) column with a 0-300 mM NaC1 gradient in 50 mM sodium acetate (pH 4.0), 4 mM 2-mercaptoethanol, 0.1% NP-40, 6 M urea also can afford some purification. Final purification of the RNase M5 activity to homogeneity is achieved by denaturing (SDS) polyacrylamide gel electrophoresis, recovering the protein according to the protocol of Hager and Burgess.~8 A single gel band of Mr 24,000 is highly active against the BL16--pre-rRNA RNP substrate; however, the number of peptide chains required to form the active enzyme is not known. Purification of Ribosomal Proteins BL16 and EL18. Ribosomal proteins BL16 and EL18 originally were purified as activities (/3) that are required for the RNase M5 reaction. When it was discovered that the/3 activities are ribosomal proteins, the following protocol was devised. It is similar to methods commonly used to purify ribosomal proteins.11 Frozen E. coil or B. subtilis cell paste (50 g) is thawed in 50 ml of buffer B [20 mM Tris-HC1 (pH 7.8), 30 mM NH4CI, 10 mM magnesium acetate, 10 mM 2-mercaptoethanol] containing 1 /xg/ml DNase I (Sigma Type II) and broken by passage through an Aminco French pressure cell at 20,000 psi. All steps in the purification schedule are carried out at or below 4 °. The lysate is diluted with 50 ml of buffer B and centrifuged for 20 min at 18,000 rpm in a Sorvall SS34 rotor. The supernatant is centrifuged for 5 hr at 45,000 rpm in a Beckman 50.2 Ti rotor, and the ribosome pellet resuspended in buffer B. The NH4CI concentration is adjusted to 1 M and the volume to 120 ml by the addition of buffer B, and the ribosomes are again pelleted. The salt-washed ribosomes are resuspended in 75 ml of buffer B. After dialysis overnight against 20 mM Tris-HC1 (pH 7.8), 10 mM NH4CI, 2 mM magnesium acetate, 10 mM 2-mercaptoethanol, 0.05% Brij 58 (or NP-40), ribosomes are dispersed by addition of urea crystals to 8.5 M. After 24 hr on ice, the urea concentration is reduced to 6.5 M by addition of 50 mM sodium phosphate (pH 6.5), and a fine precipitate is removed by centrifugation for 5 min at 18,000 rpm in a Sorvall SS34 rotor. Approximately 25,000 A260units of supernatant is loaded onto a 500-ml phosphocellulose column equilibrated with 50 mM sodium phosphate (pH 6.5), 6 M urea, 2 mM methylamine, 0.05% NP-40 and washed with several hundred milliliters of the same buffer. Proteins are eluted with a 0-700 is D. A. Hager and R. R. Burgess, Anal. Biochem. 109, 76 (1980).
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PROCESSINGOF RIBOSOMALRNAs
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mM NH4CI gradient in the same buffer; the activities that complement the RNase M5 reaction elute at about 300 mM. Peak activity-containing fractions are pooled, dialyzed against 50 mM acetic acid (pH 3), 0.1 mM EDTA, 0.1% NP-40, 30 mM NH4C1, loaded onto a 15-ml phosphocellulose column in the same buffer, and eluted with a 0.03-1.2 M gradient of NH4CI in that buffer. Both BL16 and EL18 elute late, at approximately 800 mM NH4CI, and are nearly pure at this stage. A third column chromatography is carried out on a 2-ml phosphocellulose column in 40 mM TrisHC1 (pH 7.4), 0.1 mM EDTA, 0.1% NP-40, 30 mM NH4CI, eluting with a 30-700 mM NH4CI gradient; the ribosomal proteins elute at approximately 400 mM. A fourth column, required only for rigorous purification of the ELI8, is the same as the third except that all buffers contain 6 M urea. The EL18 elutes at approximately 150 mM in the 20-400 mM NH4CI gradient. Pooled fractions are dialyzed against buffer B containing 0.1% NP-40 and stored as aliquots frozen at -70 °. Acknowledgments The authors' research is supported by National Institutes of Health Grant GM34527to N.R.P. We thank David Smithfor artwork.
