[29]
ASSAYOF E. coli rRNA MATURATIONin Vitro
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[29] P r e p a r a t i o n o f E x t r a c t s a n d A s s a y o f R i b o s o m a l R N A M a t u r a t i o n in E s c h e r i c h i a coli
By
ANAND
K.
SRIVASTAVA
and
DAVID
SCHLESSINGER
Like nearly all prokaryotic RNA species rRNA is made initially as a larger precursor form. 1 In Escherichia coli, the 30 S pre-rRNA transcript includes a leader sequence, spacer sequences containing tRNA species, and a trailer sequence in addition to the 16, 23, and 5 S rRNAs (Fig. 1A). Precursor sequences at the 5' and 3' ends of 16 and 23 S rRNAs contain complementary regions which form strong base-paired stems enclosing the mature species (Fig. 1B,C). In wild-type cells, rRNA transcripts are initially cleaved at these stems by well-defined enzymes, especially RNase III, to yield the various precursor species (pre-16, pre-23, and pre-5 S rRNA) which are subsequently processed to mature RNAs by the action of additional enzymes. J To study further the formation of mature termini in detail, appropriate substrates and sensitive assays are necessary. Here we describe (1) assay for maturation of termini and source of substrate; (2) preparation of extract and subcellular fractions; (3) preparation of substrates; and (4) maturation assay in vitro. Assays for Maturation of Termini Nuclease protection assays and primer-extension methods are adaptable as quantitative assays of rRNA processing. They have been used to detect both the steady-state levels of pre-rRNA and mature RNA species in vivo and the course of processing reactions in vitro using restriction fragments of the rrnB operon complementary to the appropriate region of rRNA as probes. Ribosomes and rRNAs from different strains of E. coli provide appropriate substrates. Strain D 10 (RNase I-; Ref. 2) is taken as a reference strain ("wild-type processing"). For studies of 23 S rRNA maturation, pre-50 S ribosomes and pre-23 S rRNA are prepared from the RNase III-deficient strain ABL1. 3 Since RNase III is indispensable for 23 S rRNA maturation, essentially all of the RNA chains containing the T. C. King, R. Sirdeshmukh, and D. Schlessinger, Microbiol. Rev. 50, 428 (1986). 2 R. F. Gesteland, J. Mol. Biol. 16, 67 (1966). 3 H. D. Robertson, E. G. Pelle, and W. H. McClain, in "Transfer RNA: Biological Aspects" (P. R. Schimmel, D. Soil, and J. N. Abelson, eds), p. 107. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1980.
METHODS IN ENZYMOLOGY, VOL. 181
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. The rrnB operon and possible base-paired stems that enclose 16 and 23 S rRNA sequences in pre-rRNA transcripts. (A) Operon drawn to scale. Mature rRNA sequence ( m ) . (B and C) Secondary structure of pre-rRNA sequence adjoining the 5' and 3' ends of 16 and 23 S rRNA. An open arrowhead (A) indicates the amount of rrnB spacer sequence included in the probe used for the nuclease protection assays (see Fig. 2). RNase III cleavages, which occur very fast in wild-type cells, are indicated by closed arrows. ( ~ , Mature terminus. Nucleotide numbers are as in J. Brosius, T. J. Dull, D. D. Sleeter, and H. F. Noller, J. Mol. Biol. 148, 107 (1981).
[29]
ASSAY OF E. coli rRNA MATURATIONin Vitro
357
23 S r R N A sequence are immature, showing no background of mature termini. N o strain with a total a b s e n c e of mature 16 S r R N A is known, p r e s u m ably b e c a u s e maturation of 16 S r R N A is required for biological function. 4,5 As a result, all assays of maturation must be p e r f o r m e d in the p r e s e n c e of an appreciable level of mature termini. The B U M M E R strain, partially deficient in 5'-end-processing for 16 S r R N A , 6 is used as a source o f p r e - 1 6 S r R N A and preribosomes. In addition, 7 - 1 0 % o f the 16 S r R N A in A B L I or D10 cells is in p r e c u r s o r form and can provide a substrate for s o m e reactions. 7 Precursor 5 S r R N A can also be accumulated in an appropriate mutant that is t e m p e r a t u r e sensitive in R N a s e E. 8 Nuclease Protection Assay
The S 1 nuclease protection assay for the analysis of r R N A processing involves the hybridization of unlabeled r R N A to a 32p-end-labeled restriction fragment of rrnB. Double-stranded D N A which is c o m p l e m e n t a r y to a sequence spanning the mature terminus can be used with denaturing conditions of hybridization, or single-stranded D N A with nondenaturing conditions. The R N A - D N A hybrids thus formed are treated with S! nuclease to digest nonhybridized sequences. The size of the resultant S 1resistant D N A fragments (protected hybrids) are then analyzed by electrophoresis in u r e a - p o l y a c r y l a m i d e gels. The design and labeling o f p r o b e depend on the R N A terminus studied as well as the purpose o f the experiment. To m e a s u r e levels of both mature and precursor termini, probes are used in excess containing sequences that border the mature terminus (i.e., hybridize to both mature and p r e c u r s o r r R N A sequences). Such probes are labeled at the end within the mature sequence, and can be used to quantitate both mature and p r e c u r s o r species, and to assay final maturation steps by the detection of the disappearance of p r e c u r s o r and the production of mature termini during the reaction. ( N o t e : Again, this a p p r o a c h is feasible only for 23 S r R N A maturation, where the substrate contains no initial level of mature r R N A termini.) The same probe is labeled at the other end (within the p r e c u r s o r sequence) in order to measure only p r e c u r s o r species. Either type of p r o b e can be used to follow the processing of the p r e c u r s o r sequences. 4 j. w. Wireman and P. S. Sypherd, Nature (London) 247, 552 (1974). 5 M. Nomura and W. A. Held, in "Ribosomes" (M. Nomura, A. Tissieres, and P. Lengyel, eds.), p. 193. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1974. 6 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). 7 T. C. King and D. Schlessinger, J. Biol. Chem. 258, 12034 (1983). 8 T. K. Misra and D. Apirion, J. Biol. Chem. 254, 11154 (1979).
358
PROCESSINGOF RIBOSOMALRNAs
[29]
The maturation of 16 S rRNA is more difficult to assay, because all the available E. coli strains contain variable levels of pre-16 S rRNA and high levels of mature 16 S rRNA. As a result, it is difficult to measure the formation of the mature product over the high background; nevertheless, it is possible to assess processing by detecting the specific precursor fragment released from pre-16 S rRNA during maturation. 6,9 Fortunately, the released fragment is stable and can be detected by nuclease protection assays using a probe labeled within the precursor sequence (though it is degraded to smaller fragments during prolonged incubations in cell extracts10). An alternative strategy is also possible, using the techniques of Sigmund et al., 11 which make it possible to discriminate plasmid-promoted 16 S rRNA even in the presence of large amounts of chromosomal 16 S product. Some of the probes used are cloned in pBR322, 7 whereas others are prepared as restriction fragments from rrnB (see below). To label the 5' end, the restriction fragments are first dephosphorylated with bacterial alkaline phosphatase and then labeled at the 5' ends with [y-3Zp]ATP and T4 polynucleotide kinase. 12 The 3'ends of restriction fragments are labeled by filling in overhanging 5' ends with the Klenow fragment of DNA polymerase I (or overhanging 3' ends with T4 DNA polymerase) and appropriate [a-32p]deoxynucleoside triphosphates.12 Labeled DNA is purified on polyacrylamide gels (50:1 acrylamide-bisacrylamide) using 0.5 × tris(hydroxymethyl)aminomethane (Tris)-borate-ethylenediaminetetraacetic acid (EDTA) buffer, as described by Maxam and Gilbert. 13The strands of the DNA fragments are then separated by heating in 45% dimethyl sulfoxide (DMSO) (v/v) for 5 min at 90° followed by rapid chilling in a dry ice-ethanol bath. Single-stranded DNA is repurified and isolated as above on a 5% polyacrylamide gel. Hybridization and SI Nuclease Digestion. Hybridization of end-labeled single-stranded DNA fragments and rRNA follows the procedure of Casey and Davidson 14(as employed in Ref. 7). Here 0.2-2.5/xg of rRNA and 3000-5000 cpm of 32p-end-labeled single-stranded probe DNA in 1 × hybridization buffer [100 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (pH 6.4), 400 mM NaC1, 1 mM EDTA] are coprecipitated with ethanol in the presence of 2/zg of yeast tRNA as carrier. 9 F. Hayes and M. Vasseur, Eur. J. Biochem. 61, 433 (1976). to A. K. Srivastava and D. Schlessinger, Nucleic Acids Res. 17, 1649 (1989). ~ C. D. Sigmund, M. Ettayebi, A. Borden, and E. A. Morgan, this series, in press. t2 T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982. ~3A. M. Maxam and W. Gilbert, this series, Vol. 65, p. 499. 14j. Casey and N. Davidson, Nucleic Acids Res. 5, 1539 (1977).
[29]
ASSAYOF E. coli rRNA MATURATIONin Vitro
359
After centrifugation, the pellet is redissolved in 20/zl of hybridization buffer and heated for 5-10 min at 75 ° to assure denaturation. The samples are quickly transferred to a water bath at 62-64 °, and hybridization is carried out for 2-3 hr. The reaction is terminated by rapid 10-fold dilution with 200/zl of ice-cold digestion buffer [280 mM NaC1, 50 mM sodium acetate (pH 4.6), and 4.5 mM zinc sulfate] containing 40 units of S1 endonuclease. After incubation for 1 hr at 37°, samples are chilled, and the S l-resistant nucleic acid is precipitated with 2.5 volumes of ethanol in the presence of 10/xg yeast tRNA at - 4 5 ° for 30 min. After centrifugation for 15 min in an Eppendorf centrifuge, the nucleic acid pellet is washed with 70% ethanol, dried, and resuspended in 10 /xl of 90% deionized formamide containing 0.05% each of the size markers xylene cyanol and bromphenol blue. Fractionation of products is usually carried out in a 45-cm-long by 0.5-mm-thick 6-8% polyacrylamide (30:1 acrylamide bisacrylamide) gel containing 8.3 M urea, 90 mM Tris-borate, and 2 mM EDTA (pH 8.0). Comments. Hybridization is carried out under denaturing conditions in a buffer (as described above) containing 80% formamide when doublestranded DNA (instead of single-stranded) is used as a probe. In each such case the temperature of hybridization should be optimized. Exonuclease VII, which hydrolyzes single-stranded DNA from both 5' and 3' termini, 15,16can be used instead of SI nuclease. This enzyme has an advantage over SI nuclease when hybrids contain a high percentage of mismatched bases or unstable AT-rich sequences that would otherwise be cleaved by SI nuclease. In such assays, the hybridization reaction is diluted with 200/xl of ice-cold buffer [10 mM Tris-HCl (pH 7.4), 30 mM KCI, 10 mM EDTA, and I0 mM 2-mercaptoethanol] containing 2 units of exonuclease VII and incubated for 1 hr at 45 °. The reaction is terminated by the addition of NaCI to 0.1 M and 2 volumes of ethanol. Subsequent treatment is similar to that described above for the S 1 nuclease protection assay. Primer-Extension Assay
The primer-extension assay essentially involves the synthesis of DNA copies of the rRNA present with reverse trancriptase. Here it is illustrated for the assay of maturation of the 5' end of 23 S rRNA. The primerextension method has recently been employed to measure the fraction of rRNA modified with a single point mutation; it is effective even on crude RNA fractions.ll This approach can be extended further to other rRNAs with appropriate primers. 15 j. W. C h a s e and C. C. Richardson, J. Biol. Chem. 249, 4545 (1974). ~6 j. W. C h a s e and C. C. Richardson, J. Biol. Chem. 249, 4553 (1974).
360
PROCESSINGOF RIBOSOMALRNAs
[29]
Method. To study the formation of the 5' end of 23 S rRNA, the primer is a single-stranded DNA containing nucleotides 3606-3513 (see Fig. 2E below). The double-stranded restriction fragment is end-labeled at nucleotide 3606. From this a single-stranded DNA is purified on a polyacrylamide gel as described above. Ribosomal RNA is annealed to a singlestranded DNA (8,000-10,000 cpm) in 20/zl of hybridization buffer under the conditions described for the S1 nuclease protection assay. After 3 hr at 64°, a temperature that optimizes RNA-DNA hybrid formation, the annealed nucleic acids are precipitated by the addition of sodium acetate, to 0.25 M, and 2.5 volumes of ethanol at -45 °. After centrifugation, the hybrids are redissolved in reverse transcriptase buffer [50 mM Tris-HCl (pH 8.3), 50 mM KCI, 6 mM MgC12, and 10 mM dithiothreitol (DTT)]. The primer is extended on the RNA template in a total reaction volume of 20 /zl with 1 mM each of dCTP, dTTP, dGTP, and dATP and 14 units of avian myeioblastosis virus (AMV) reverse transcriptase for 1 hr at 41 °. The solution is brought to 25 mM EDTA and 0.2 N NaOH and incubated for an additional 30 min at 30°. Twelve microliters of 1 M Tris (pH 7.5) and 5 /~g of yeast tRNA are added, and the volume is brought to 200/zl with water. The nucleic acids are then extracted with phenol and precipitated with ethanol at -45 °. The precipitate is washed with 70% ethanol, dried, and redissolved in 90% formamide (v/v) and fractionated in an 8% ureapolyacrylamide gel as described above.
Preparation of Extract and Subcellular Fractions The maturation of precursor 16 S rRNA is independent of RNase III cleavage, 7 but this cleavage is obligate for the maturation of 23 S rRNA. ~7 Both RNase III and other enzymes required for the maturation of rRNA can be supplied by using a "ribosome wash" (see below) from a wild-type strain of E. coli, namely, D10. Buffers and Solutions
Buffer I: 10 mM Tris-HCl (pH 7.4), 5 mM MgCI2, and 2 mM CaCI2 Buffer II: 10 mM Tris-HCl (pH 7.8), 60 mM NH4CI, 10 mM MgCI2, and 10 mM 2-mercaptoethanol Buffer III: I0 mM Tris-HCl (pH 7.8), 1 M NH4CI, 10 mM MgCI2, and 10 mM 2-mercaptoethanol Buffer IV: 20 mM Tris-HCl (pH 7.6), 50 mM NH4CI, 5 mM 2-mercaptoethanol, 0.1 mM EDTA, and 10% glycerol 17T. C. King,R. Sirdeshmukh,and D. Schlessinger,Proc. Natl. Acad. Sci. U.S.A. 81, 185 (1984).
[29]
ASSAYOF E. coli rRNA MATURATIONin Vitro
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Buffer V: 10 mM Tris-HCI (pH 7.4), 10 mM magnesium acetate, and 6 mM 2-mercaptoethanoi Buffer VI: 10 mM Tris-HCl (pH 7.4), 60 mM NH4CI, 1.0 mM magnesium acetate, and 6 mM 2-mercaptoethanol Buffer VII: 120 mM Tris-HCl (pH 7.8), 4 mM EDTA, and 20 mg/ml lysozyme Buffer VIII: 6 mM Tris-HCl (pH 7.8), 90 mM NH4CI, 35 mM magnesium acetate, 1.5% Brij 35, and 0.1% sodium deoxycholate Buffer IX: 10 mM Tris-HC1 (pH 7.8), 1 M NHaCI, 40 mM magnesium acetate, 2 mM EDTA, and 10 mM 2-mercaptoethanol Buffer X: 0.2 M sodium acetate (pH 5.5), 0.1 M NaCI, 1.0 mM EDTA, and 0.5% sodium dodecyl sulfate (SDS) Growth o f Cells. Escherichia coli strain D10 is grown at 37° in Luria broth to an optical density of 0.55-0.65 at 550 nm. The cells are harvested on ice, washed once with ice-cold buffer I, and stored at - 7 0 ° until use. The processing-deficient strains ABL1 and BUMMER are grown in broth at 30°. All subsequent steps are carried out at 0-4 °, unless otherwise stated. Crude Extract. Frozen cells (2.5 g) are ground with 5 g of alumina powder (twice the wet weight of cells) in a prechilled mortar for 4-5 min (until the mixture forms a thin homogeneous paste). The cell paste is suspended in 7.5 ml of buffer II (3 ml per gram of cells). The suspension is centrifuged for 10 min at 12,000 g in a Sorvall SS34 rotor to remove the alumina. The supernatant (crude extract) is decanted and recentrifuged for 20 min at 30,000 g. SIO0 and Ribosome Wash. The crude extract is layered on 30% sucrose in buffer II (1 ml for each 5 ml of the extract) and centrifuged at 105,000 g for 3 hr in a Beckman Type 65 fixed-angle rotor. The upper twothirds of the supernatant is collected (S100 fraction; source of soluble factors), dialyzed for 12 hr against buffer V, and centrifuged at 10,000 g for 10 min to discard any precipitate. The clear supernatant is stored frozen at - 2 0 °. The ribosome pellet is resuspended and gently homogenized in 5 ml of buffer III and left for 1 hr at 0°. The suspension is again centrifuged at 105,000 g for 3 hr, and the upper 85% of the supernatant phase is recovered as the "ribosome wash." The volume of ribosome wash is measured, and proteins are precipitated by the addition of powdered ammonium sulfate (0.4 g/ml). The solution is slowly stirred for 10 min, and the precipitated proteins are then recovered by centrifugation for 10 min at 12,000 g in a Sorvall SS34 rotor. The pellet is dissolved in 1 ml of buffer IV and dialyzed against the same buffer for 12 hr. The protein solution is centrifuged 5 min in an Eppendorf centrifuge, the precipitate discarded, and the clear solution stored frozen at - 2 0 °.
362
PROCESSING OF RIBOSOMAL R N A s
[29]
Preparation of Substrates
Ribosomes. Frozen cells are ground with alumina and the crude extract made as described before. The concentration of ammonium chloride in the crude extract is brought to I M, and the mixture is centrifuged through a layer of 30% sucrose (1 ml for every 5 ml of extract) in the same buffer, at 105,000 g for 4 hr. The supernatant is discarded and the ribosomal pellet is suspended in buffer VI. The ribosomal solution (1 ml) is carefully layered on top of a 38-ml linear gradient of 10-30% sucrose in buffer V1 and centrifuged at 50,000 g for 16 hr. Fractions are collected, and the positions of the ribosomal subunits are localized by measuring their optical density at 260 nm. Peak fractions containing 50 and 30 S ribosomal subunits are pooled separately. The magnesium concentration is raised to 10 mM, and the ribosomes are stored in 50% glycerol at - 2 0 °. Polysomes. The preparation is modified from Ref. 18. Escherichia coli cells are grown to an absorbance of 0.5 at 550 nm in 1 liter of broth containing [3H]uridine (0.5 mCi). The growing cells are quickly chilled by pouring over crushed ice in the presence of chloramphenicol (100/zg/ml) and then harvested by centrifugation at 30,000 g for 15 min. The pellet is washed with ice-cold buffer I (20 ml) and recentrifuged. Cells are dispersed in 0.4 ml of buffer VII containing lysozyme (added just before use) and incubated at 10°. After 10-15 rain of incubation, 1.0 ml of buffer VIII at 10° is added, and the incubation is continued until a clear lysate forms. The mixture is then treated for 5 min at I0° with 50/zg/ml RNase-free DNase I. The lysate is clarified by centrifugation at 12,000 g for 3 min. The supernatant is collected, and the pellet is reextracted with 0.5 ml of buffer VIII. The combined supernatants are cleared of any remaining cell debris by centrifugation at 5,000 g for 10 min. The total supernatant (-1.5 ml) is then layered on a 38-ml linear gradient of I0-30% sucrose in buffer IX and centrifuged at 35,000 g for 15 hr. In a similar gradient 30, 50, and 70 S ribosomes are centrifuged as markers. Monosomes and polysomes of different sizes are localized in gradient fractions by measuring the absorbance at 260 nm and monitoring the radioactivity. Fractions containing polysomes are pooled and stored in 50% glycerol at - 2 0 °. The largest polysomes are used for maturation studies. (Note: It has been systematically verified by zonal sedimentation in sucrose gradients that the polysomes are quantitatively converted to monosomes by mild treatment with pancreatic RNase.) Extraction ofrRNA. Ribosomal RNA is isolated from ribosomes and polysomes by phenol extraction. Solutions of ribosomes or polysomes are ~8T. Girb6s, B. Cabrer, and J. Modolell, this series, Vol. 59, p. 353.
[29]
ASSAYOF E. coli rRNA MATURATIONin Vitro
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used at concentrations less than 200-300 A260 units/ml, and 0.1 volume of 10% SDS and 0.2 volume of 2% bentonite suspension are added. The mixture is vortexed for 2-3 min with an equal volume of phenol. The aqueous layer is separated by centrifugation, and phenol extraction is repeated 3 more times. The aqueous layer is extracted once with phenolchloroform. One-third volume of 1 M sodium acetate (pH 4.6) is added, and the RNA is then precipitated with 2.5 volumes of ethanol at - 2 0 ° for at least 1 hr. The RNA precipitate is pelleted by centrifugation at 10,000 g for 15 min, dried, dissolved in hybridization buffer, and stored at - 2 0 °. Isolation o f Total R N A f r o m Cells. Total RNA from bacterial cells is isolated to assess rRNA termini present in vioo. Eschericha coli cells are grown in 10-ml cultures, harvested, and washed with buffer 1. Cells are suspended in buffer X (500/xl) and extracted with an equal volume of phenol at 50°. Extraction of the aqueous phase is repeated 3 times at room temperature, and 2 volumes of cold ethanol is then added, after which the mixture is placed at - 2 0 °. Following centrifugation, the RNA pellet is washed with 70% ethanol, dried, and dissolved in hybridization buffer. Maturation Assays in Vitro The substrates for final maturation, if prepared from an RNase IIIcontaining strain, are already cleaved in the double-stranded stems as shown in Fig. 1. In this case the pre-16 S rRNA has 115 precursor nucleotides at its 5' end and 33 at its 3' end (Fig. 1B); the pre-23 S rRNA has 3 or 7 extra 5' nucleotides (cleavage at M + 3 and M + 7) and 7 to 9 extra 3' nucleotides (Fig. 1C). An RNA species 4 nucleotides shorter than the mature 5' terminus of 23 rRNA has also been reported. ~9If substrates are instead prepared from the RNase IlI-deficient strain the precursor segments are much longer. Another mutant provides an additional source of precursors to study the maturation of the 5' terminus of 16 S rRNA: the BUMMER strain contains 50% of its 16 S rRNA chains with an extra 66 nucleotides. The substrates of choice for 23 S rRNA maturation are ribosomes from strain ABL1 and, because RNase III is indispensable for processing, extracts of strain D10 (RNase III positive). In contrast, 16 S rRNA is matured by endonucleolytic cleavages at the mature termini even in the absence of any RNase III cleavages. 7,~° As a result, the substrates of choice are ribosomes from the RNase III-deficient or BUMMER strain and extracts of either the RNase III-negative strain or strain DI0. It must be kept in mind that efficient maturation has been demont9 R. Sirdeshmukh,M. Krych, and D. Schlessinger, Nucleic Acids Res. 13, 1185 (1985).
364
PROCESSING OF RIBOSOMAL R N A s
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FIG. 2. Hybridization probe for nuclease protection and primer-extension assays. (A and C) Probes to study the 5' end of 16 and 23 S rRNA, respectively. (B and D) Probes for the 3' end of 16 and 23 S rRNA. (E) Primer DNA to study 5' terminus of 23 S rRNA. Asterisks mark the labeled end of each probe. Nucleotide numbers are as in J. Brosius, T. J. Dull, D. D. Sleeter, and H. F. Noller, J. Mol. Biol. 148, 107 (1981).
strated only with polysomes (or ribosomes and protein synthetic conditions). 2° This dictates the conditions of many of the assays.
23 S rRNA Probes. The single-stranded probe for the 5' terminus of 23 S rRNA (from an HpalI restriction fragment of a cloned portion of rrnB) extends from nucleotide 3404 to 3606 (and contains 105 nucleotides of mature 23 S rRNA sequence and 97 nucleotides of adjacent spacer) (Fig. 2C). The total probe of 331 base pairs (bp) contains 129 nucleotides of pBR322 sequence 5' to the spacer rDNA sequence. 7 The 3'-terminal probe is 2o A. K. Srivastava and D. Schlessinger, Proc. Natl. Acad. Sci. U.S.A. 74, 7144 (1988).
[29]
ASSAYOF E. coli rRNA MATURATIONin Vitro
365
complementary to 29 nucleotides of mature 23 S rRNA and 94 nucleotides of adjacent precursor sequence (nucleotides 6374-6497) (Fig. 2D), as well as 104 nucleotides of pBR322 sequence 3' to the precursor sequence. 7 Assay. One microgram of ribosomes or polysomes from the RNase Ill-deficient strain ABL1, in a total volume of 50/.d of 10 mM Tris-HCl (pH 7.6), containing 180 mM NH4CI, 8 mM MgCI2, 5 mM 2-mercaptoethanol, 0. I mM EDTA, and 10% glycerol, is incubated with or without 5/zl of ribosome wash from wild-type strain D10 for 60-120 min at 37°. After the reaction, one-third volume of 1 M sodium acetate (pH 4.6), 5/zl of 10% SDS, and I0/zg of yeast tRNA are added, and the mixture is extracted with an equal volume of phenol-chloroform. The aqueous phase is separated from the phenol layer by centrifugation for 3 min in an Eppendorf centrifuge. RNA is precipitated from the aqueous phase with addition of 2.5 volumes of cold ethanol at -45 ° for at least 30 min. After centrifugation for 15 min at 4° in an Eppendorf centrifuge, the RNA pellet is washed with cold 70% ethanol, dried, and dissolved in 20/~1 of hybridization buffer. Between 5 and 10 p.1 of RNA solution is used for each assay. The remainder is stored at - 2 0 ° until used. 16 S rRNA Probes. Figure 2A shows a typical probe for the study of maturation of the 5' terminus, extending from nucleotide 1353 to 1540 and containing 23 nucleotides of mature rRNA and 165 of precursor sequence (a DdeI restriction fragment of rrnB). For the study of the 3'-terminal maturation, DNA complementary to nucleotides 2959-3156 is used (an HaelII restriction fragment of the cloned 16 S rRNA); it contains 98 nucleotides of precursor and 100 nucleotides of mature sequence, as well as 143 nucleotides of pBR322 sequence distal to the mature 16 S rRNA portion (Fig. 2B). Assay. Five micrograms of ribosomes from the BUMMER or ABL1 strain of E. coli is incubated with or without 5/zl of ribosome wash from wild-type strain D10 in a total volume of 50/zl containing 25 mM Tris-HCl (pH 7.8), 100 mM NH4CI, 10 mM MgCI2, and 6 mM 2-mercaptoethanol. After incubation for 30 min at 37°, the RNA is extracted and dissolved as described for 23 S rRNA. 5 S rRNA
Pre-5 S rRNA from the RNase E temperature-sensitive strain grown at the nonpermissive temperature has 85 precursor nucleotides at its 5' end and extra 3'-nucleotides extending to the transcription termination point. 8 RNase E cleavage of this RNA produces a species which retains 3 extra
366
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[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.
ASSAYOF E. coli rRNA MATURATIONin Vitro
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[29] P r e p a r a t i o n o f E x t r a c t s a n d A s s a y o f R i b o s o m a l R N A M a t u r a t i o n in E s c h e r i c h i a coli
By
ANAND
K.
SRIVASTAVA
and
DAVID
SCHLESSINGER
Like nearly all prokaryotic RNA species rRNA is made initially as a larger precursor form. 1 In Escherichia coli, the 30 S pre-rRNA transcript includes a leader sequence, spacer sequences containing tRNA species, and a trailer sequence in addition to the 16, 23, and 5 S rRNAs (Fig. 1A). Precursor sequences at the 5' and 3' ends of 16 and 23 S rRNAs contain complementary regions which form strong base-paired stems enclosing the mature species (Fig. 1B,C). In wild-type cells, rRNA transcripts are initially cleaved at these stems by well-defined enzymes, especially RNase III, to yield the various precursor species (pre-16, pre-23, and pre-5 S rRNA) which are subsequently processed to mature RNAs by the action of additional enzymes. J To study further the formation of mature termini in detail, appropriate substrates and sensitive assays are necessary. Here we describe (1) assay for maturation of termini and source of substrate; (2) preparation of extract and subcellular fractions; (3) preparation of substrates; and (4) maturation assay in vitro. Assays for Maturation of Termini Nuclease protection assays and primer-extension methods are adaptable as quantitative assays of rRNA processing. They have been used to detect both the steady-state levels of pre-rRNA and mature RNA species in vivo and the course of processing reactions in vitro using restriction fragments of the rrnB operon complementary to the appropriate region of rRNA as probes. Ribosomes and rRNAs from different strains of E. coli provide appropriate substrates. Strain D 10 (RNase I-; Ref. 2) is taken as a reference strain ("wild-type processing"). For studies of 23 S rRNA maturation, pre-50 S ribosomes and pre-23 S rRNA are prepared from the RNase III-deficient strain ABL1. 3 Since RNase III is indispensable for 23 S rRNA maturation, essentially all of the RNA chains containing the T. C. King, R. Sirdeshmukh, and D. Schlessinger, Microbiol. Rev. 50, 428 (1986). 2 R. F. Gesteland, J. Mol. Biol. 16, 67 (1966). 3 H. D. Robertson, E. G. Pelle, and W. H. McClain, in "Transfer RNA: Biological Aspects" (P. R. Schimmel, D. Soil, and J. N. Abelson, eds), p. 107. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1980.
METHODS IN ENZYMOLOGY, VOL. 181
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
A PI P7 I III
165
tRNA
II ~'0()0
23S
5S
li
il
30;0
4 0 '0 0
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NUCL[OTIDES
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UA
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C
-.~
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A
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A U'G
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u
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o
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A A
~
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•
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5S
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c
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c
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u
~:oo
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o
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UU-A
u
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~-U 3 4 0 0 - c -G U-A A-U - 6 4 5 0 G A G-C
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G
U-A C-G A U A A A~u G 5' UGCUC UUUAAAA. UAAAUCAGAACGC A
FIG. 1. The rrnB operon and possible base-paired stems that enclose 16 and 23 S rRNA sequences in pre-rRNA transcripts. (A) Operon drawn to scale. Mature rRNA sequence ( m ) . (B and C) Secondary structure of pre-rRNA sequence adjoining the 5' and 3' ends of 16 and 23 S rRNA. An open arrowhead (A) indicates the amount of rrnB spacer sequence included in the probe used for the nuclease protection assays (see Fig. 2). RNase III cleavages, which occur very fast in wild-type cells, are indicated by closed arrows. ( ~ , Mature terminus. Nucleotide numbers are as in J. Brosius, T. J. Dull, D. D. Sleeter, and H. F. Noller, J. Mol. Biol. 148, 107 (1981).
[29]
ASSAY OF E. coli rRNA MATURATIONin Vitro
357
23 S r R N A sequence are immature, showing no background of mature termini. N o strain with a total a b s e n c e of mature 16 S r R N A is known, p r e s u m ably b e c a u s e maturation of 16 S r R N A is required for biological function. 4,5 As a result, all assays of maturation must be p e r f o r m e d in the p r e s e n c e of an appreciable level of mature termini. The B U M M E R strain, partially deficient in 5'-end-processing for 16 S r R N A , 6 is used as a source o f p r e - 1 6 S r R N A and preribosomes. In addition, 7 - 1 0 % o f the 16 S r R N A in A B L I or D10 cells is in p r e c u r s o r form and can provide a substrate for s o m e reactions. 7 Precursor 5 S r R N A can also be accumulated in an appropriate mutant that is t e m p e r a t u r e sensitive in R N a s e E. 8 Nuclease Protection Assay
The S 1 nuclease protection assay for the analysis of r R N A processing involves the hybridization of unlabeled r R N A to a 32p-end-labeled restriction fragment of rrnB. Double-stranded D N A which is c o m p l e m e n t a r y to a sequence spanning the mature terminus can be used with denaturing conditions of hybridization, or single-stranded D N A with nondenaturing conditions. The R N A - D N A hybrids thus formed are treated with S! nuclease to digest nonhybridized sequences. The size of the resultant S 1resistant D N A fragments (protected hybrids) are then analyzed by electrophoresis in u r e a - p o l y a c r y l a m i d e gels. The design and labeling o f p r o b e depend on the R N A terminus studied as well as the purpose o f the experiment. To m e a s u r e levels of both mature and precursor termini, probes are used in excess containing sequences that border the mature terminus (i.e., hybridize to both mature and p r e c u r s o r r R N A sequences). Such probes are labeled at the end within the mature sequence, and can be used to quantitate both mature and p r e c u r s o r species, and to assay final maturation steps by the detection of the disappearance of p r e c u r s o r and the production of mature termini during the reaction. ( N o t e : Again, this a p p r o a c h is feasible only for 23 S r R N A maturation, where the substrate contains no initial level of mature r R N A termini.) The same probe is labeled at the other end (within the p r e c u r s o r sequence) in order to measure only p r e c u r s o r species. Either type of p r o b e can be used to follow the processing of the p r e c u r s o r sequences. 4 j. w. Wireman and P. S. Sypherd, Nature (London) 247, 552 (1974). 5 M. Nomura and W. A. Held, in "Ribosomes" (M. Nomura, A. Tissieres, and P. Lengyel, eds.), p. 193. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1974. 6 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). 7 T. C. King and D. Schlessinger, J. Biol. Chem. 258, 12034 (1983). 8 T. K. Misra and D. Apirion, J. Biol. Chem. 254, 11154 (1979).
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PROCESSINGOF RIBOSOMALRNAs
[29]
The maturation of 16 S rRNA is more difficult to assay, because all the available E. coli strains contain variable levels of pre-16 S rRNA and high levels of mature 16 S rRNA. As a result, it is difficult to measure the formation of the mature product over the high background; nevertheless, it is possible to assess processing by detecting the specific precursor fragment released from pre-16 S rRNA during maturation. 6,9 Fortunately, the released fragment is stable and can be detected by nuclease protection assays using a probe labeled within the precursor sequence (though it is degraded to smaller fragments during prolonged incubations in cell extracts10). An alternative strategy is also possible, using the techniques of Sigmund et al., 11 which make it possible to discriminate plasmid-promoted 16 S rRNA even in the presence of large amounts of chromosomal 16 S product. Some of the probes used are cloned in pBR322, 7 whereas others are prepared as restriction fragments from rrnB (see below). To label the 5' end, the restriction fragments are first dephosphorylated with bacterial alkaline phosphatase and then labeled at the 5' ends with [y-3Zp]ATP and T4 polynucleotide kinase. 12 The 3'ends of restriction fragments are labeled by filling in overhanging 5' ends with the Klenow fragment of DNA polymerase I (or overhanging 3' ends with T4 DNA polymerase) and appropriate [a-32p]deoxynucleoside triphosphates.12 Labeled DNA is purified on polyacrylamide gels (50:1 acrylamide-bisacrylamide) using 0.5 × tris(hydroxymethyl)aminomethane (Tris)-borate-ethylenediaminetetraacetic acid (EDTA) buffer, as described by Maxam and Gilbert. 13The strands of the DNA fragments are then separated by heating in 45% dimethyl sulfoxide (DMSO) (v/v) for 5 min at 90° followed by rapid chilling in a dry ice-ethanol bath. Single-stranded DNA is repurified and isolated as above on a 5% polyacrylamide gel. Hybridization and SI Nuclease Digestion. Hybridization of end-labeled single-stranded DNA fragments and rRNA follows the procedure of Casey and Davidson 14(as employed in Ref. 7). Here 0.2-2.5/xg of rRNA and 3000-5000 cpm of 32p-end-labeled single-stranded probe DNA in 1 × hybridization buffer [100 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (pH 6.4), 400 mM NaC1, 1 mM EDTA] are coprecipitated with ethanol in the presence of 2/zg of yeast tRNA as carrier. 9 F. Hayes and M. Vasseur, Eur. J. Biochem. 61, 433 (1976). to A. K. Srivastava and D. Schlessinger, Nucleic Acids Res. 17, 1649 (1989). ~ C. D. Sigmund, M. Ettayebi, A. Borden, and E. A. Morgan, this series, in press. t2 T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982. ~3A. M. Maxam and W. Gilbert, this series, Vol. 65, p. 499. 14j. Casey and N. Davidson, Nucleic Acids Res. 5, 1539 (1977).
[29]
ASSAYOF E. coli rRNA MATURATIONin Vitro
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After centrifugation, the pellet is redissolved in 20/zl of hybridization buffer and heated for 5-10 min at 75 ° to assure denaturation. The samples are quickly transferred to a water bath at 62-64 °, and hybridization is carried out for 2-3 hr. The reaction is terminated by rapid 10-fold dilution with 200/zl of ice-cold digestion buffer [280 mM NaC1, 50 mM sodium acetate (pH 4.6), and 4.5 mM zinc sulfate] containing 40 units of S1 endonuclease. After incubation for 1 hr at 37°, samples are chilled, and the S l-resistant nucleic acid is precipitated with 2.5 volumes of ethanol in the presence of 10/xg yeast tRNA at - 4 5 ° for 30 min. After centrifugation for 15 min in an Eppendorf centrifuge, the nucleic acid pellet is washed with 70% ethanol, dried, and resuspended in 10 /xl of 90% deionized formamide containing 0.05% each of the size markers xylene cyanol and bromphenol blue. Fractionation of products is usually carried out in a 45-cm-long by 0.5-mm-thick 6-8% polyacrylamide (30:1 acrylamide bisacrylamide) gel containing 8.3 M urea, 90 mM Tris-borate, and 2 mM EDTA (pH 8.0). Comments. Hybridization is carried out under denaturing conditions in a buffer (as described above) containing 80% formamide when doublestranded DNA (instead of single-stranded) is used as a probe. In each such case the temperature of hybridization should be optimized. Exonuclease VII, which hydrolyzes single-stranded DNA from both 5' and 3' termini, 15,16can be used instead of SI nuclease. This enzyme has an advantage over SI nuclease when hybrids contain a high percentage of mismatched bases or unstable AT-rich sequences that would otherwise be cleaved by SI nuclease. In such assays, the hybridization reaction is diluted with 200/xl of ice-cold buffer [10 mM Tris-HCl (pH 7.4), 30 mM KCI, 10 mM EDTA, and I0 mM 2-mercaptoethanol] containing 2 units of exonuclease VII and incubated for 1 hr at 45 °. The reaction is terminated by the addition of NaCI to 0.1 M and 2 volumes of ethanol. Subsequent treatment is similar to that described above for the S 1 nuclease protection assay. Primer-Extension Assay
The primer-extension assay essentially involves the synthesis of DNA copies of the rRNA present with reverse trancriptase. Here it is illustrated for the assay of maturation of the 5' end of 23 S rRNA. The primerextension method has recently been employed to measure the fraction of rRNA modified with a single point mutation; it is effective even on crude RNA fractions.ll This approach can be extended further to other rRNAs with appropriate primers. 15 j. W. C h a s e and C. C. Richardson, J. Biol. Chem. 249, 4545 (1974). ~6 j. W. C h a s e and C. C. Richardson, J. Biol. Chem. 249, 4553 (1974).
360
PROCESSINGOF RIBOSOMALRNAs
[29]
Method. To study the formation of the 5' end of 23 S rRNA, the primer is a single-stranded DNA containing nucleotides 3606-3513 (see Fig. 2E below). The double-stranded restriction fragment is end-labeled at nucleotide 3606. From this a single-stranded DNA is purified on a polyacrylamide gel as described above. Ribosomal RNA is annealed to a singlestranded DNA (8,000-10,000 cpm) in 20/zl of hybridization buffer under the conditions described for the S1 nuclease protection assay. After 3 hr at 64°, a temperature that optimizes RNA-DNA hybrid formation, the annealed nucleic acids are precipitated by the addition of sodium acetate, to 0.25 M, and 2.5 volumes of ethanol at -45 °. After centrifugation, the hybrids are redissolved in reverse transcriptase buffer [50 mM Tris-HCl (pH 8.3), 50 mM KCI, 6 mM MgC12, and 10 mM dithiothreitol (DTT)]. The primer is extended on the RNA template in a total reaction volume of 20 /zl with 1 mM each of dCTP, dTTP, dGTP, and dATP and 14 units of avian myeioblastosis virus (AMV) reverse transcriptase for 1 hr at 41 °. The solution is brought to 25 mM EDTA and 0.2 N NaOH and incubated for an additional 30 min at 30°. Twelve microliters of 1 M Tris (pH 7.5) and 5 /~g of yeast tRNA are added, and the volume is brought to 200/zl with water. The nucleic acids are then extracted with phenol and precipitated with ethanol at -45 °. The precipitate is washed with 70% ethanol, dried, and redissolved in 90% formamide (v/v) and fractionated in an 8% ureapolyacrylamide gel as described above.
Preparation of Extract and Subcellular Fractions The maturation of precursor 16 S rRNA is independent of RNase III cleavage, 7 but this cleavage is obligate for the maturation of 23 S rRNA. ~7 Both RNase III and other enzymes required for the maturation of rRNA can be supplied by using a "ribosome wash" (see below) from a wild-type strain of E. coli, namely, D10. Buffers and Solutions
Buffer I: 10 mM Tris-HCl (pH 7.4), 5 mM MgCI2, and 2 mM CaCI2 Buffer II: 10 mM Tris-HCl (pH 7.8), 60 mM NH4CI, 10 mM MgCI2, and 10 mM 2-mercaptoethanol Buffer III: I0 mM Tris-HCl (pH 7.8), 1 M NH4CI, 10 mM MgCI2, and 10 mM 2-mercaptoethanol Buffer IV: 20 mM Tris-HCl (pH 7.6), 50 mM NH4CI, 5 mM 2-mercaptoethanol, 0.1 mM EDTA, and 10% glycerol 17T. C. King,R. Sirdeshmukh,and D. Schlessinger,Proc. Natl. Acad. Sci. U.S.A. 81, 185 (1984).
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ASSAYOF E. coli rRNA MATURATIONin Vitro
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Buffer V: 10 mM Tris-HCI (pH 7.4), 10 mM magnesium acetate, and 6 mM 2-mercaptoethanoi Buffer VI: 10 mM Tris-HCl (pH 7.4), 60 mM NH4CI, 1.0 mM magnesium acetate, and 6 mM 2-mercaptoethanol Buffer VII: 120 mM Tris-HCl (pH 7.8), 4 mM EDTA, and 20 mg/ml lysozyme Buffer VIII: 6 mM Tris-HCl (pH 7.8), 90 mM NH4CI, 35 mM magnesium acetate, 1.5% Brij 35, and 0.1% sodium deoxycholate Buffer IX: 10 mM Tris-HC1 (pH 7.8), 1 M NHaCI, 40 mM magnesium acetate, 2 mM EDTA, and 10 mM 2-mercaptoethanol Buffer X: 0.2 M sodium acetate (pH 5.5), 0.1 M NaCI, 1.0 mM EDTA, and 0.5% sodium dodecyl sulfate (SDS) Growth o f Cells. Escherichia coli strain D10 is grown at 37° in Luria broth to an optical density of 0.55-0.65 at 550 nm. The cells are harvested on ice, washed once with ice-cold buffer I, and stored at - 7 0 ° until use. The processing-deficient strains ABL1 and BUMMER are grown in broth at 30°. All subsequent steps are carried out at 0-4 °, unless otherwise stated. Crude Extract. Frozen cells (2.5 g) are ground with 5 g of alumina powder (twice the wet weight of cells) in a prechilled mortar for 4-5 min (until the mixture forms a thin homogeneous paste). The cell paste is suspended in 7.5 ml of buffer II (3 ml per gram of cells). The suspension is centrifuged for 10 min at 12,000 g in a Sorvall SS34 rotor to remove the alumina. The supernatant (crude extract) is decanted and recentrifuged for 20 min at 30,000 g. SIO0 and Ribosome Wash. The crude extract is layered on 30% sucrose in buffer II (1 ml for each 5 ml of the extract) and centrifuged at 105,000 g for 3 hr in a Beckman Type 65 fixed-angle rotor. The upper twothirds of the supernatant is collected (S100 fraction; source of soluble factors), dialyzed for 12 hr against buffer V, and centrifuged at 10,000 g for 10 min to discard any precipitate. The clear supernatant is stored frozen at - 2 0 °. The ribosome pellet is resuspended and gently homogenized in 5 ml of buffer III and left for 1 hr at 0°. The suspension is again centrifuged at 105,000 g for 3 hr, and the upper 85% of the supernatant phase is recovered as the "ribosome wash." The volume of ribosome wash is measured, and proteins are precipitated by the addition of powdered ammonium sulfate (0.4 g/ml). The solution is slowly stirred for 10 min, and the precipitated proteins are then recovered by centrifugation for 10 min at 12,000 g in a Sorvall SS34 rotor. The pellet is dissolved in 1 ml of buffer IV and dialyzed against the same buffer for 12 hr. The protein solution is centrifuged 5 min in an Eppendorf centrifuge, the precipitate discarded, and the clear solution stored frozen at - 2 0 °.
362
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Preparation of Substrates
Ribosomes. Frozen cells are ground with alumina and the crude extract made as described before. The concentration of ammonium chloride in the crude extract is brought to I M, and the mixture is centrifuged through a layer of 30% sucrose (1 ml for every 5 ml of extract) in the same buffer, at 105,000 g for 4 hr. The supernatant is discarded and the ribosomal pellet is suspended in buffer VI. The ribosomal solution (1 ml) is carefully layered on top of a 38-ml linear gradient of 10-30% sucrose in buffer V1 and centrifuged at 50,000 g for 16 hr. Fractions are collected, and the positions of the ribosomal subunits are localized by measuring their optical density at 260 nm. Peak fractions containing 50 and 30 S ribosomal subunits are pooled separately. The magnesium concentration is raised to 10 mM, and the ribosomes are stored in 50% glycerol at - 2 0 °. Polysomes. The preparation is modified from Ref. 18. Escherichia coli cells are grown to an absorbance of 0.5 at 550 nm in 1 liter of broth containing [3H]uridine (0.5 mCi). The growing cells are quickly chilled by pouring over crushed ice in the presence of chloramphenicol (100/zg/ml) and then harvested by centrifugation at 30,000 g for 15 min. The pellet is washed with ice-cold buffer I (20 ml) and recentrifuged. Cells are dispersed in 0.4 ml of buffer VII containing lysozyme (added just before use) and incubated at 10°. After 10-15 rain of incubation, 1.0 ml of buffer VIII at 10° is added, and the incubation is continued until a clear lysate forms. The mixture is then treated for 5 min at I0° with 50/zg/ml RNase-free DNase I. The lysate is clarified by centrifugation at 12,000 g for 3 min. The supernatant is collected, and the pellet is reextracted with 0.5 ml of buffer VIII. The combined supernatants are cleared of any remaining cell debris by centrifugation at 5,000 g for 10 min. The total supernatant (-1.5 ml) is then layered on a 38-ml linear gradient of I0-30% sucrose in buffer IX and centrifuged at 35,000 g for 15 hr. In a similar gradient 30, 50, and 70 S ribosomes are centrifuged as markers. Monosomes and polysomes of different sizes are localized in gradient fractions by measuring the absorbance at 260 nm and monitoring the radioactivity. Fractions containing polysomes are pooled and stored in 50% glycerol at - 2 0 °. The largest polysomes are used for maturation studies. (Note: It has been systematically verified by zonal sedimentation in sucrose gradients that the polysomes are quantitatively converted to monosomes by mild treatment with pancreatic RNase.) Extraction ofrRNA. Ribosomal RNA is isolated from ribosomes and polysomes by phenol extraction. Solutions of ribosomes or polysomes are ~8T. Girb6s, B. Cabrer, and J. Modolell, this series, Vol. 59, p. 353.
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ASSAYOF E. coli rRNA MATURATIONin Vitro
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used at concentrations less than 200-300 A260 units/ml, and 0.1 volume of 10% SDS and 0.2 volume of 2% bentonite suspension are added. The mixture is vortexed for 2-3 min with an equal volume of phenol. The aqueous layer is separated by centrifugation, and phenol extraction is repeated 3 more times. The aqueous layer is extracted once with phenolchloroform. One-third volume of 1 M sodium acetate (pH 4.6) is added, and the RNA is then precipitated with 2.5 volumes of ethanol at - 2 0 ° for at least 1 hr. The RNA precipitate is pelleted by centrifugation at 10,000 g for 15 min, dried, dissolved in hybridization buffer, and stored at - 2 0 °. Isolation o f Total R N A f r o m Cells. Total RNA from bacterial cells is isolated to assess rRNA termini present in vioo. Eschericha coli cells are grown in 10-ml cultures, harvested, and washed with buffer 1. Cells are suspended in buffer X (500/xl) and extracted with an equal volume of phenol at 50°. Extraction of the aqueous phase is repeated 3 times at room temperature, and 2 volumes of cold ethanol is then added, after which the mixture is placed at - 2 0 °. Following centrifugation, the RNA pellet is washed with 70% ethanol, dried, and dissolved in hybridization buffer. Maturation Assays in Vitro The substrates for final maturation, if prepared from an RNase IIIcontaining strain, are already cleaved in the double-stranded stems as shown in Fig. 1. In this case the pre-16 S rRNA has 115 precursor nucleotides at its 5' end and 33 at its 3' end (Fig. 1B); the pre-23 S rRNA has 3 or 7 extra 5' nucleotides (cleavage at M + 3 and M + 7) and 7 to 9 extra 3' nucleotides (Fig. 1C). An RNA species 4 nucleotides shorter than the mature 5' terminus of 23 rRNA has also been reported. ~9If substrates are instead prepared from the RNase IlI-deficient strain the precursor segments are much longer. Another mutant provides an additional source of precursors to study the maturation of the 5' terminus of 16 S rRNA: the BUMMER strain contains 50% of its 16 S rRNA chains with an extra 66 nucleotides. The substrates of choice for 23 S rRNA maturation are ribosomes from strain ABL1 and, because RNase III is indispensable for processing, extracts of strain D10 (RNase III positive). In contrast, 16 S rRNA is matured by endonucleolytic cleavages at the mature termini even in the absence of any RNase III cleavages. 7,~° As a result, the substrates of choice are ribosomes from the RNase III-deficient or BUMMER strain and extracts of either the RNase III-negative strain or strain DI0. It must be kept in mind that efficient maturation has been demont9 R. Sirdeshmukh,M. Krych, and D. Schlessinger, Nucleic Acids Res. 13, 1185 (1985).
364
PROCESSING OF RIBOSOMAL R N A s
P2
A
5'U5~7} r~/.
Gene Probe
[29]
16s r R N A ~
I
*
(3059) 3'
B
Gene ~ 6 s
Probe
5'(3230)
rRNA~,~
LpBR
I
5'(3499)
(3306) 3'
C
Oene
~ 2 3 5
Probe
I
L pBR (6403)3'
D
Gene Probe
~/~.23SrRNA ~
*
rRNA~
*
5' (6495) ",~ rRNA
I
( pBR _/--Y
E
(3306) 3' Gene
Primer
~
5' (3499} ~23SrRNA~
,
FIG. 2. Hybridization probe for nuclease protection and primer-extension assays. (A and C) Probes to study the 5' end of 16 and 23 S rRNA, respectively. (B and D) Probes for the 3' end of 16 and 23 S rRNA. (E) Primer DNA to study 5' terminus of 23 S rRNA. Asterisks mark the labeled end of each probe. Nucleotide numbers are as in J. Brosius, T. J. Dull, D. D. Sleeter, and H. F. Noller, J. Mol. Biol. 148, 107 (1981).
strated only with polysomes (or ribosomes and protein synthetic conditions). 2° This dictates the conditions of many of the assays.
23 S rRNA Probes. The single-stranded probe for the 5' terminus of 23 S rRNA (from an HpalI restriction fragment of a cloned portion of rrnB) extends from nucleotide 3404 to 3606 (and contains 105 nucleotides of mature 23 S rRNA sequence and 97 nucleotides of adjacent spacer) (Fig. 2C). The total probe of 331 base pairs (bp) contains 129 nucleotides of pBR322 sequence 5' to the spacer rDNA sequence. 7 The 3'-terminal probe is 2o A. K. Srivastava and D. Schlessinger, Proc. Natl. Acad. Sci. U.S.A. 74, 7144 (1988).
[29]
ASSAYOF E. coli rRNA MATURATIONin Vitro
365
complementary to 29 nucleotides of mature 23 S rRNA and 94 nucleotides of adjacent precursor sequence (nucleotides 6374-6497) (Fig. 2D), as well as 104 nucleotides of pBR322 sequence 3' to the precursor sequence. 7 Assay. One microgram of ribosomes or polysomes from the RNase Ill-deficient strain ABL1, in a total volume of 50/.d of 10 mM Tris-HCl (pH 7.6), containing 180 mM NH4CI, 8 mM MgCI2, 5 mM 2-mercaptoethanol, 0. I mM EDTA, and 10% glycerol, is incubated with or without 5/zl of ribosome wash from wild-type strain D10 for 60-120 min at 37°. After the reaction, one-third volume of 1 M sodium acetate (pH 4.6), 5/zl of 10% SDS, and I0/zg of yeast tRNA are added, and the mixture is extracted with an equal volume of phenol-chloroform. The aqueous phase is separated from the phenol layer by centrifugation for 3 min in an Eppendorf centrifuge. RNA is precipitated from the aqueous phase with addition of 2.5 volumes of cold ethanol at -45 ° for at least 30 min. After centrifugation for 15 min at 4° in an Eppendorf centrifuge, the RNA pellet is washed with cold 70% ethanol, dried, and dissolved in 20/~1 of hybridization buffer. Between 5 and 10 p.1 of RNA solution is used for each assay. The remainder is stored at - 2 0 ° until used. 16 S rRNA Probes. Figure 2A shows a typical probe for the study of maturation of the 5' terminus, extending from nucleotide 1353 to 1540 and containing 23 nucleotides of mature rRNA and 165 of precursor sequence (a DdeI restriction fragment of rrnB). For the study of the 3'-terminal maturation, DNA complementary to nucleotides 2959-3156 is used (an HaelII restriction fragment of the cloned 16 S rRNA); it contains 98 nucleotides of precursor and 100 nucleotides of mature sequence, as well as 143 nucleotides of pBR322 sequence distal to the mature 16 S rRNA portion (Fig. 2B). Assay. Five micrograms of ribosomes from the BUMMER or ABL1 strain of E. coli is incubated with or without 5/zl of ribosome wash from wild-type strain D10 in a total volume of 50/zl containing 25 mM Tris-HCl (pH 7.8), 100 mM NH4CI, 10 mM MgCI2, and 6 mM 2-mercaptoethanol. After incubation for 30 min at 37°, the RNA is extracted and dissolved as described for 23 S rRNA. 5 S rRNA
Pre-5 S rRNA from the RNase E temperature-sensitive strain grown at the nonpermissive temperature has 85 precursor nucleotides at its 5' end and extra 3'-nucleotides extending to the transcription termination point. 8 RNase E cleavage of this RNA produces a species which retains 3 extra
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
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