E. coli RNAase P has a required RNA component in vivo

Cell. Vol. 19, 881-887,

April 1980,

Copyright

0 1980

by MIT

E. coli RNAase P Has a Required in Vivo Ryszarcl Kole, Madeline F. Baer, Benjamin C. Stark* and Sidney Altmant Department of Biology Yale University New Haven, Connecticut 06520

Results

Summary RNAase P has been partially purified from three thermosensitive strains of E. coli and the thermal inactivation characteristics of each preparation have been determined. The RNAase P preparations from two of these mutant strains, ts241 and ts709, and the wild-type strain have been separated into RNA and protein components. Various mixtures of the reconstituted components have been checked in vitro for complementation of their thermal sensitivity properties. The protein component of RNAase P from ts241 and the RNA component of RNAase P from ts709, respectively, account for the thermal sensitivity of the RNAase P from the two strains. The amount of the RNA component of RNAase P is lower in ts709 than in ts241 or the wild-type parent, 4273. RNAase P partially purified from a revertant of the third mutant strain, A49, which maps at or near the ts241 mutation, has an altered charge when compared to the RNAase P from the parent strain, BF265. We conclude that mutations which affect either the protein or RNA component of RNAase P can confer thermal sensitivity on the enzyme both in vivo and in vitro. Introduction The essential role of RNAase P in E. coli tRNA biosynthesis has been demonstrated with the use of temperature-sensitive mutants (Schedl and Primakoff, 1973; Sakano et al., 1974; reviewed by Altman, 1978). These mutant strains accumulate precursors to all tRNAs at restrictive temperatures (Schedl, Primakoff and Roberts, 1974; lkemura et al., 1975). Crude extracts of these mutant strains manifest thermosensitive RNAase P activity in vitro. RNAase P activity in vitro depends upon the presence of both RNA and protein components (Stark et al., 1978; Kole and Altman, 1979) but the need for an essential RNA component of the enzyme in vivo has not yet been demonstrated. From complementation experiments in vitro and other biochemical data, however, we are now able to conclude that the thermosensitive properties of RNAase P from mutants of E. coli can be ascribed to alterations in either the RNA or protein component of RNAase P purified from individual mutant strains. * Present address: Department of Biology, Indiana ington. Indiana 47401. t To whom reprint requests should be addressed.

RNA Component

University.

Bloom-

Partially purified RNAase P preparations from three thermosensitive strains of E. coli and their parents (Table 1) have been checked for their thermosensitivity in vitro. The results show (Table 2), as expected, that RNAase P preparations from strains ts241 and ts709 are temperature-sensitive compared with the RNAase P from their parent strain, 4273. The specific activity of RNAase P from ts709 is considerably lower than that of either 4273 or ts241. The mutant strain A49 has even lower specific activity of RNAase P than does ts709. In our thermal inactivation experiments, RNAase P from A49 does not exhibit temperature sensitivity when compared with its wild-type parent strain, BF265 (Table 2). However, the specific activity of this mutant enzyme is only about 1% of wild-type. The low specific activity of RNAase P from A49 is apparent even in crude extracts assayed at 3O’C. It is possible that the assembly of mature enzyme in this strain is temperature-sensitive but that the resultant enzymatic activity, when purified from crude cell extracts, is normal with respect to its thermal sensitivity. The results shown in Table 2 suggest, but do not prove, that some structural component of RNAase P is affected by the ts241 and ts709 mutations. We present additional evidence below, indicating the likelihood that the A49 site also affects a structural component of RNAase P. Complementation Studies in Vitro To demonstrate that the ts241 and ts709 mutations separately determine protein and RNA components of RNAase P, we undertook a series of complementation experiments in vitro. These experiments were carried out using separated RNA and protein components of RNAase P partially purified from the appropriate strains. The separated RNA and protein components of RNAase P are inactive but can be combined together in vitro to reconstitute active enzyme (Kale and Altman, 1979). Hybrid enzymes reconstituted in this way were tested for thermal sensitivity properties by preincubating the enzyme to be tested at either 47’ or 30°C and then assaying for residual RNAase P activity at 37C. (The degree of inactivation by preincubation at 47’ is greater than observed after preincubation at 42’C: the latter temperature was used for the experiments summarized in Table 2.) Any complementation in vitro which overcomes thermal sensitivity of mutant enzyme components and which occurred in reconstituted, hybrid enzymes is observed easily when the preincubation temperature is 47°C. The results show that the RNAase P preparations from ts241 and ts709 are temperature-sensitive when compared with RNAase P from the parent strain, 4273 (Table 3, first three lines). Virtually no activity is detectable in the mutant enzymes after preincubation at

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Table

1. E. coli Strains

Used

in Studies

of RNAase

Parental Strain for Mutant or Revertant

Mutant or Revertant Strain

Approximate Map Location of Mutantsa

A4gb

BF265b

80-82’

ts709=

4273’

69-70’

ts241 c

4273

80-82’

A49-3+*

A49

ND

a Y. Shimura, D. Apirion, B. J. Bachmann communications. b Schedl and Primakoff (1973). ’ Sakano et al. (1974). d Received from W. H. McClain, University (ND) Not determined.

Table 2. Some Properties with Temperature-Sensitive

Mutant

Strain

and

K. B. Low,

from

SD

Sensitivity

of Native

% Residual (47”/3O”C)

Enzyme

and Reconstituted

RNAase

P

Activity Number

12

6

ts241 a

1

6

ts70ga

0

9

23

9

Protein

RNA

4273

MREGOO

ts241

MREGOO

5

12

ts709

MREGOO

20

9

4273

4273

30

3

4273

ts241

55

3

4273

ts709

t13b

9

ts709

ts241

58

3

of Trials

E. coli Strains

Number Trials

A49

0.015

1 .oo

0.17

5

ts241

0.72

0.78

0.16

4

ts709

0.19

0.69

0.14

8

,

personal

Relative HalfLife at 42’C of Mutant Strain RNAase Pb Average

3. Thermal

4273a

of Wisconsin.

of RNAase P Isolated RNAase P Function

Relative Specific Activity of Mutant Strain RNAase Pa

Table

P

of

a Parent wild-type strains are listed in Table 1. Enzymes were prepared and assayed as described in Experimental Procedures. Assays of parent enzymes generally used 20-200 ng of protein, whereas assays of mutant extracts used proportionally more, according to the specific activities shown in the table. b Thermal inactivation half-lives were calculated relative to those measured for the parent strains under the same conditions. Calculations of thermal inactivation half-lives, including standard deviations (SD), are described in Experimental Procedures. ‘L.’

47°C whereas about 12% activity (compared with that measured after preincubation at 30”) is left in the parent enzyme. When the separated protein components of RNAase P from the mutant strains are combined with RNA taken from RNAase P extracted from MREGOO, a conveniently available wild-type strain, to form hybrid RNAase P, only one of the hybrid enzymes is now temperature-sensitive: the one made with ts241 protein (Table 3, lines 4-6). The temperature sensitivity of the latter hybrid is about the same as that of native RNAase P extracted from ts241. The ts709 protein-MREGOO RNA hybrid enzyme is not temperature-sensitive, and we have also found that its specific activity is greater than that of native ts709 enzyme. These results support the hypothesis that the temperature-sensitive nature of RNAase P from ts241 is due to the protein component of the enzyme, and that the RNA component of ts709 is responsible for the temperature-sensitive nature of that enzyme. The hypothesis is supported further by experiments summarized in Table 3, lines 7-l 0.

a Native enzyme, wild-type or mutant, exposed to 7 M urea and then dialyzed to remove the urea, subsequently shows about 50% of the original activity; the recovered activity has the same thermal inactivation characteristics as enzyme untreated with urea. b The activity of this reconstituted enzyme was always very low, so ratios of activity of treated enzyme are somewhat unreliable. In one measurement a residual relative activity of 13% was measured. In eight other measurements only trace activity was recovered after preincubation at 30°, and no activity was recovered after preincubation at 47’C. Appropriate samples of RNAase P were assayed after preincubation at 30’ or 47°C as described in Experimental Procedures. The % relative residual activity is the ratio of the activity of enzyme treated at 47’ to the activity of the enzyme treated at 30°C. Reconstitutions were carried out as described in Experimental Procedures. Reconstitution experiments with RNA from 4273, ts241 and ts709 yielded enzyme with generally lower specific activities than other combinations tried. Furthermore, the absolute amounts of RNA we obtained from these strains in the preparative procedures were also lower than those obtained from comparable amounts of MREGOO cells. Both these observations may be due to the fact that the 4273 series is RNAase I+, and thus the RNA in these strains suffers more degradation during our enzyme purification than does the comparable RNA from MREGOO. which is RNAase I-.

If hybrid enzymes are made with a wild-type protein component of RNAase P (from 4273) and RNA components from various strains, only the hybrid made with RNA from ts709 remains temperature-sensitive. Finally, and of greatest significance, we note that the hybrid made with its protein component from ts709 and its RdA component from ts241 has wild-type thermal sensitivity characteristics as we would now expect. This result clearly shows that the protein from ts709 and the RNA from ts241 can complement in vitro. Conversely, we can conclude that the RNA from ts709 and the protein from ts241 are responsible for the temperature-sensitive phenotype of the RNAase P extracted from each strain, respectively. The data listed in Table 3 do not prove that the ts241 and ts709 loci code directly for the protein and RNA components of RNAase P, but they do indicate that these loci must code for some products which affect the nature of the components of RNAase P.

RNAase 883

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In Vivo

ts709 RNA To demonstrate further that the ts709 locus affects the RNA component of RNAase P, we examined and compared electrophoretic separations of RNA extracted from 4273 and its two mutant derivatives, ts241 and ts709 (Figure 1). The most significant apparent difference is the decrease in the amount of the RNA species labeled Ml shown in the ts709 lane of Figure 1. We have observed that the RNA extracted from purified RNAase P preparations has an RNAase Tl fingerprint (Figure 2) very similar to that of an RNA species called band IX RNA (Ikemura and Dahlberg, 1973). When the RNA extracted from RNAase P is run in polyacrylamide gels, it splits into two bands, one having the mobility of Ml RNA and the other having the mobility of M2 RNA (see Figure 1). RNAase Tl fingerprints (generated by the methods of Sanger, Brownlee and Barrell, 1965) of Ml and M2 RNA prepared as described in the legend to Figure 1 are very similar to each other and to that of band IX RNA (not shown). The fingerprints of Ml and M2 RNA do show some small difference in the yield of certain oligonucleotides, indicating that they may be very similar but not identical. In fact, when the same species are analyzed using a different fingerprinting technique (Platt and Yanofsky, 1975) which resolves larger oligonucleotides better in the second dimension (homochromatography), clearer differences are visible. In this case, RNAase Tl fingerprints of Ml and M2 RNAs (Figures 3A and 38) or pancreatic RNAase A fingerprints (Figures 3C and 3D) show nonidentical patterns of large oligonucleotides, mainly in the upper left portions of the fingerprints under comparison. The RNAase Tl fingerprint of M2 RNA is identical to that of an’ RNA called 1 OS RNA (Ray and Apirion, 1979). However, we have estimated the size of M2 RNA, using denaturing glyoxal gels (McMaster and Carmichael, 1977) as 360 nucleotides in contrast to a much larger estimate for 1 OS RNA given by Ray and Apirion (1979). We cannot say whether Ml or M2 RNA is absolutely identical to band IX RNA. This is because of the absence of detailed nucleotide sequence analysis of band IX RNA and differences in strains used to prepare the RNAs. We have found, for example, that when Ml RNA is rerun in 4.8% polyacrylamide gels, it does not migrate as a pure species but rather splits into two bands, one having its original mobility and a second having the mobility of M2 RNA. M2 RNA, however, reruns as only one band with its original mobility. In our previous work (Stark et al., 1978; Kole and Altman, 1979) we have described the RNA extracted from RNAase P as M2 RNA although we now know it is a mixture of Ml and M2 RNAs, each of which has a distinct fingerprint. However, it is clear from the results shown in Figure 1 that the amount of Ml RNA is severely depleted in ts709 at 30°C. In

4273

TS 241

TS 709

5S-bpb 4s.

Figure 1 Electrophoretic 4273, ts241 and ts709

Separation

of RNAs

Extracted

from E. coli

32P-labeled RNA was prepared, extracted and electrophoresed as described in Experimental Procedures. Electrophoresis was carried out in a 4.8% acrylamide gel with the current set at 20 mA for 16 hr at 4°C. The positions of the dye markers, xylene cyanol and bromophenol blue, and the various RNAs are shown in the figure.

Cdl 884

Figure 2. Fingerprints of M2 Extracted from RNAase P

RNA and

RNA

The figure shows separation of oligonucleotides by electrophoresis on cellulose acetate (first dimension, right to left) and electrophoresis on Whatman DEAE-paper (second dimension, top to bottom). Cligonucleotides were generated using RNAase Tl and fingerprinting was carried out as described by Sanger et al. (1965). (Left) M2 RNA (E. coli MREGOO). The electrophoretic mobility of this RNA is very close to that of band IX RNA. (Right) Mixture of Ml and M2 RNAs extracted from RNAase P (E. coli MREGOO). The arrows indicate pGp visible in the original films.

Figure 3. Fingerprints (E. coli 4273)

of Ml

and M2

RNAs

The figure shows separation of oligonucleotides by electrophoresis on cellulose acetate (first dimension) and by homochromatography on polyethyleneimine plates (second dimension) as indicated in (6). The species fingerprinted are the ones shown labeled in Figure 1, and the fingerprinting was carried out as described by Platt and Yanofsky (1975). (A) Ml RNA: RNAase Tl fingerprint. (6) M2 RNA: RNAase Tl finaerprint. (C) Ml RNA: pancreatic RNAase-A fingerprint. (D) M2 RNA: pancreatic RNAase A fingerprint.

addition, M2 RNA isolated from ts709 (as shown in Figure 1) has the same fingerprint as M2 RNA from 4273, its parent strain, and ts241. Ml RNA from ts241 has the same fingerprint as Ml RNA from 4273. These results indicate that normal production of wildtype Ml RNA may be necessary for thermal stability of RNAase P. It is interesting to note that the RNA component of RNAase P extracted from ts709 func-

tioned very inefficiently in the reconstitution mixtures. All these data suggest that the ts709 locus affects the RNA moiety of RNAase P. A49 and A49-3+ Protein The ts241 and A49 mutants appear to map at or near the same position in E. coli (Table 1). We have examined the electrophoretic properties of a revertant

RNAase 885

P Has

a Required

RNA

Component

In Vivo

of A49 called A49-3+ to investigate whether the functionally revertant RNAase P molecules have an altered charge compared with the original enzyme. When RNAase P partially purified from A49-3+ is electrophoresed in an isoelectric focusing gel, its measured pl, 4.9, is different by approximately 0.3 pH units from that of the original parent of A49, namely BF265 (Figure 4). We have also observed small but reproducible differences in the relative mobilities of these enzymes in nondenaturing gel systems run under a variety of conditions. RNAase P from A49 also exhibits differences in electrophoretic mobility and pl compared with those of BF265, but to a lesser extent than RNAase P from A49-3+ (Stark, 1977). The changes in electrophoretic properties indicate that the mutant A49 and A49-3+ enzymes are more acidic than the wild-type RNAase P. One interpretation *of these results is that the protein component of the mutant enzymes has an altered charge compared with that of the wild-type enzyme. We have determined, in separate experiments, that RNAase P from A49 and ts709 have the same gel filtration properties in Sephadex G-200 as their wild-type parent enzymes (Stark, 19771, so the differences in their electrophoretic properties cannot be due to differences in size. Discussion We have shown previously that RNAase P requires both a protein and an RNA component for activity in vitro (Stark et al., 1978; Kole and Altman, 1979). It was conceivable, although unlikely, that some ill-defined factor had led to the requirement for RNA in the RNAase P reaction in vitro and that the RNA component would not be required in vivo. We have now demonstrated that two different mutations which affect RNAase P function in vivo also affect either the protein or RNA component of RNAase P in vitro. Furthermore, when RNA from ts241 RNAase P (which has an altered protein component) is reconstituted with protein from ts709 RNAase P (which has an altered RNA component), the resultant hybrid enzyme has wild-type thermal inactivation properties. These data demonstrate that a mutation which makes RNAase P thermosensitive in vivo also confers thermosensitivity on RNAase P in vitro, and that this defective phenotype can be overcome by the addition of RNA from strains which are not defective in this component of the enzyme. Furthermore, we have presented evidence that the amount of the Ml RNA in the ts709 mutant strain is altered end that protein from the A49-3+ strain (a revertant of the A49 strain which maps at or near the ts241 site) is also altered. These data do not prove that the various mutant sites code directly for RNA or protein components of RNAase P, but do indicate that they must, at least, code for some factors that alter in vivo the RNA and protein components, respectively, of RNAase P.

Figure 4. Altered Mobility Isoelectric Focusing Gels

of RNAase

P from

Strain

A49-3+

on

RNAase P from strains RF265 and A49-3+ were run simultaneously in identical pH 3.5-10 isoelectric focusing gels. They were fractionated and eluted identically, so that fractions of the same number have the same mobility. Fractions are numbered in order of increasing acidity (with low numbers at the basic ends of the gels). The fractions were assayed using tRNATY’ precursor as substrate; the mobilities of the RNAase P products of this substrate (“tRNA” and the 5’ fragment) are indicated. Only fractions around the peak of RNAase P activity are shown.

The reason for the change in the amount of ts709 Ml RNA is unclear. There is precedent for considering that a single base change could lead to temperaturesensitive function and underproduction of stable, functional RNA, namely temperature-sensitive mutants of tRNATY’su$ (Smith et al., 1970). These mutant tRNAs are made in greatly reduced amounts compared with their parent and are temperature-sensitive for suppressor function. The phenotype of these mutants, as may be the case for ts709, would presumably be due to the destruction of some hydrogen bonding scheme in RNA caused by a single nucleotide change, and this in turn would lead to greater susceptibility of that RNA to nonspecific, degradative nuclease action. Another possibility is that the ts709 mutation affects interaction with an RNA processing enzyme, pieventing mature Ml RNA from being made from a larger Precursor. Without mature Ml RNA, RNAase P might be temperature-sensitive although it might contain the

Cell 886

precursor to Ml RNA. Finally, the ts709 mutation may affect a nucleotide-modifying enzyme such as a methylase. Without particular modifications appearing in a mature Ml RNA, RNAase P may become temperaturesensitive, just as the properties of ribosomal RNA can be affected by the absence of certain nucleotide modifications (for review see Jaskunas, Nomura and Davies, 1974). The specific activities of RNAase P isolated from ts709 and especially from A49 are reduced compared with those of their parent strains. While the explanations proposed above may account for the phenotype of ts709 RNAase P and the apparent decrease of Ml RNA in that strain, different reasoning may be necessary to explain the apparent lack of thermal sensitivity of A49 and its low specific activity. One possibility is that the accumulation of the protein component or assembly of the enzyme is temperature-sensitive due to a mutation in the protein component. In contrast to RNAase P from ts709, RNAase P from A49 may behave as does wild-type enzyme once the enzyme complex has been formed and is purified. These hypotheses can be checked by determining the absolute amounts of the protein and RNA components of RNAase P found in A49 and by studying their assembly and stability in vitro. RNAase P-like activities are present in various eucaryotic organisms (Altman, 1978). The RNA component of RNAase P in E. coli may be required for enzyme function, perhaps through participation in substrate recognition. If so, we would expect such a component to be present in eucaryotic RNAase P. However, the relative sizes and proportions of the RNA and protein components may not be the same in eucaryotes as they are in E. coli, by analogy to the variations in ribosome structure in different organisms and organelles (reviewed by Nomura, Tissibres and Lengyel, 1974). Experimental

Procedures

Bacterial and Bacteriophage Strains E. coli 4273, ts241 and ts709 were received from Y. Shimura and H. Ozeki (Kyoto). E. coli BF265 and A49 were received from P. Schedl (Stanford), and E. coli A49-3+, a non-temperature-sensitive revertant of A49, was received from W. McClain (Madison). The approximate map positions of the mutations which confer temperature sensitivity are listed in Table 1. Other bacterial and bacteriophage strains used in the preparation of RNAase P or its substrates have been described previously (Robertson, Altman and Smith, 1972). Isoelectric Focusing of RNAase P Gels (85 mm x 6 mm cylinders) were made according to the methods of Fawcett (1968): no urea was used: the acrylamide concentration was 6% and that of bisacrylamide was 0.16%. The ampholyte was LKB Ampholine (pH 3.5-10). Samples containing FiNAase P were concentrated and dialyzed versus bufferG [20 mM Tris-HCI (pH 7.6), 0.5 M NH&I, 15 mM Mg(OAc&. 6 mM 2-mercaptoethanol, 5% sucrose] and loaded in a volume of about 75 ~1. Sample volumes were identical in cases where the mobility of samples was being compared. The gels were run at 4°C. After the run, gels were sliced (into ten equal portions), homogenized and eluted as described (Stark, 1977).

In some experiments, proteins parallel with RNAase P samples of pl versus mobility. Position determined by staining gels with Steck and Wallach, 1971).

of known isoelectric pH were run in in order to construct standard curves of focused standard proteins was Coomassie brilliant blue (Fairbanks,

Heat Inactivation Experiments For the experiments reported in Table 2, RNAase P purified through the DEAE-Sephadex stage was used (Robertson et al., 1972). Wildtype and mutant enzymes were compared using enzyme preparations made simultaneously from the various strains starting from the same amount of frozen cells. RNAase P to be used in these experiments was in buffer G. Standard RNAase P reaction mixtures were made up, and a given volume of RNAase P was added in buffer G. If wildtype and mutant enzyme were being compared, the reaction mixtures were incubated for various times at 42’C, whereupon substrate was added and incubation was continued for an additional 1 O-20 min at 37OC. Assay mixtures were then analyzed and quantitated in the usual manner. The natural logarithm of RNAase P activity was plotted versus incubation time at 42X Half-lives were calculated from the slope of a straight line fit through the data points by the method of least squares. Thus the half-life determinations are based on the assumption that heat inactivation of RNAase P is a first-order process. The correlation coefficient for the fit of the data points to a straight line was at least 0.9 in all cases, except for the data for A49, in which case it was 0.71. The low specific activity of RNAase P from A49 (see text and Table 2) made quantitation of enzymatic activity difficult and accounts in part for the low correlation coefficient measured for A49. Complementation Experiments For the experiments described in Table 3, RNAase P was first purified from 100 g of frozen cells through the G-200 Sephadex steps as outlined by Stark et al. (1978). The separated RNA component was also isolated as described by Kole and Altman (I 979) and the protein component by chromatography on a urea-CM-Sephadex column as described, except that the column was eluted with 0.5 M NaCI. Reconstitution in vitro was performed as described by Kole and Altman (1979). 50 ~1 of protein solution (7 ag) and 10 pI(3 pg) of RNA were used in all experiments. To check for RNAase P thermal sensitivity, reconstituted native RNAase P was dissolved in 100 pl final volume of the standard reaction mixture and preincubated for 1 hr at 30” or 47’C. The precursor tRNATY’ substrate was then added and a further incubation was carried out for 30 min at 37°C. The amounts of enzyme used were chosen so that no more than 50% of substrate was cleaved during the assay. The reactions were quantitated by counting appropriate slices from gels as described by Bothwell and Altman (1975). The final concentration of NH&l in the assay mixtures could range between 0.1 and 0.25 M without any effect on the thermal inactivation properties of RNAase P. Preparation of “P-Labeled RNA from E. coli 4273, ts241 and ts709 Overnight cultures of 4273, ts241 and ts709 grown at 30°C in LP medium (Smith et al., 1970) were diluted 10 fold into 5 ml of fresh medium. After 1 hr of further growth, 1.5 mCi of 32P04-3 were added to each culture, and growth continued for another 150 min. Equal volumes of phenol were added to each culture. Further steps in the extraction and electrophoresis of the labeled RNAs were carried out as described by Robertson et al. (1972) for the preparation of tRNATY’ precursor, with the exception that electrophoresis was performed in 4.8% polyacrylamide gels. Acknowledgments We are indebted to Isabel Pinto and Lois Atkins for excellent technical assistance and to Dr. A. Korner for help with the manuscript. This work was supported by grants from the USPHS and NSF to S.A. The costs of publication of this article were defrayed in part by the

RNAase 887

P Has

a Required

RNA

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In Viva

payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact. Received

December

21, 1979

References Altman, S. (1978). Transfer RNA biosynthesis. In international Review of Biochemistry, 17, B.F.C. Clark, ed. (Baltimore: University Park Press), pp. 19-44. Bothwell, A. L. M. and Altman, properties of an endoribonuclease Biol. Chem. 250, 1451-l 459.

S. (1975) isolated

Fairbanks, G., Steck, T. L. and Wallach, retie analysis of the major polypeptides membrane. Biochemistry , 2606-2617.

Partial purification and from human KB cells. J.

D. F. H. (1971). of the human

Fawcett, J. S. (1968). Isoelectric fractionation amide gels. FEBS Letters 7, 81-82.

Electrophoerythrocyte

of proteins

in polyacryl-

lkemura, T. and Dahlberg, J. E. (1973). Small ribonucleic Escherichia coli. J. Biol. Chem. 248, 5024-5032. Ikemura. T.. Shimura, Y., Sakano. H. and Ozeki, H. (1975). molecules of Escherichia coli transfer RNAs accumulated perature-sensitive mutant. J. Mol. Biol. 96, 69-86.

acids

of

Precursor in a tem-

Jaskunas. S. R.. Nomura, M. and Davies, J. (1974). Genetics of bacterial ribosomes. In Ribosomes, M. Nomura, A. Tissieres and P. Lengyel, eds. (New York: Cold Spring Harbor Laboratory), pp. 333368. Kale, A. and Altman, S. (1979). Reconstitution from inactive RNA and protein. Proc. Nat. Acad. 3799.

of RNase P activity Sci. USA 76, 3795-

McMaster, G. K. and Carmichael, G. G. (1977). Analysis of singleand double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc. Nat. Acad. Sci. USA 74, 4835-4838. Nomura, M., Tissieres, A. and Lengyel, P., eds. (1974). (New York: Cold Spring Harbor Laboratory).

Ribosomes

Platt. T. and Yanofsky. C. (1975). An intercistronic region and ribosome binding site in bacterial messenger RNA. Proc. Nat. Acad. Sci. USA 72, 2399-2403. Ray, B. K. and Apirion, D. (1979). Characterization new stable RNA molecule from Escherichia coli. 174, 25-32. Robertson, properties a tyrosine 5243-5251.

of 10s RNA: a Mol. Gen. Genet.

H., Altman, S. and Smith, J. D. (1972) Purification and of a specific Escherichia coli ribonuclease which cleaves transfer ribonucleic acid precursor. J. Biol. Chem. 247,

Sakano, H., Yamada, S.. Shimura, Y. and Ozeki. H. (1974). Temperature sensitive mutants of Escherichia coli for tRNA synthesis. Nucl. Acids Res. I, 355-371. Sanger. F., Brownlee, G. G. and Barrell. B. G. (1965). A two-dimensional fractionation procedure for radioactive nucleotides. J. Mol. Biol. 13, 373-398. Schedl, P. and Primakoff, P. (1973). Mutants of Escherichia coli thermosensitive for the synthesis of transfer RNA. Proc. Nat. Acad. Sci. USA 70, 2091-2905. Schedl, P.. Primakoff, coli tRNA precursors.

P. and Roberts, J. (1974). Processing Brookhaven Symp. Biol. 26, 53-76.

Smith, J. D., Barnett. L., Brenner. S. and Russell, R. L. (1970). mutant tyrosine transfer ribonucleic acids. J. Mol. Biol. 54, l-l Stark, B. C. (1977). Further purification ase P from Escherichia coli. Ph.D. thesis, Connecticut.

and properties Yale University,

of E. More 4.

of ribonucleNew Haven,

Stark, B. C., Kale. R., Bowman, E. J. and Altman, S. (1978). Ribonuclease P: an enzyme with an essential RNA component. Proc. Nat. Acad. Sci. USA 75, 3717-3721.