Cleavage of tRNA precursors by the RNA subunit of E. coli ribonuclease P (M1 RNA) is influenced by 3′-proximal CCA in the substrates

Cell, Vol. 36, 219-224,

August

1964, Copynght

Q 1964 by MIT

0092.8674/64/060219-06

$02.00/O

Cleavage of tRNA Precursors by the RNA Subunit of E. coli Ribonuclease P (Ml RNA) Is Influenced by 3’9Proximal CCA in the Substrates Cecilia Guerrier-Takada,* Sidney Ahman*

William H. McClain,+ and

* Department of Biology Yale University New Haven, Connecticut 06520 + Department of Bacteriology University of Wisconsin Madison, Wisconsin 53706

Summary tRNA precursor molecules that contain the CCA sequence found at the 3’ termini of all mature tRNAs are cleaved in vitro more readily by Ml RNA, the catalytic subunit of E. coli RNAase P, than precursors that lack this sequence. The sensitivity to the CCA sequence is not apparent when precursors are cleaved by the reconstituted RNAase P holoenzyme that contains both Ml RNA and the protein subunit. These results have been obtained with monomeric precursor molecules encoded by the E. coli and human chromosomes and with three dimeric precursor molecules encoded by the bacteriophage T4 genome. The data are in agreement with previous results concerning T4 tRNA biosynthesis in vivo and show that the CCA sequence is important for the processing of precursors to tRNAs. Introduction Ribonuclease P is the endoribonuclease that cleaves extra nucleotides from the 5’ termini of tRNA precursor molecules to generate the correct 5’ termini of mature tRNAs (for review see Altman et al., 1982). Recently it has been shown that the RNA subunit (Ml RNA) alone of this enzyme from E. coli can cleave tRNA precursor molecules in vitro and that the protein subunit acts as a cofactor that can both enhance the rate of the cleavage reaction and modify the substrate specificity of the RNA-protein enzyme complex (Guerrier-Takada et al., 1983; Guerrier-Takada and Altman, 1984). Accordingly, we have embarked on a study of the features of substrates that influence the ability of Ml RNA to catalyze the reaction. We examined the effect of the presence or absence of the CCA sequence at the 3’ termini of tRNA precursor molecules on the ability of Ml RNA to cleave these molecules in vitro. Our data were obtained with an E. coli tRNA precursor (with and without an enzymatically altered 3’ terminus) and with the precursor to human tRNAFMe’, and with three bacteriophage T4encoded dimeric tRNA precursor molecules, in which some of the mature tRNA sequences include the CCA sequence and some do not. With the T4 substrates we found that Ml RNA acting alone in vitro mimics exactly the requirement for the presence of the CCA sequence for cleavage by RNAase P to proceed in vivo. These results and others with the E. coli and human tRNA precursor molecule demonstrate the importance of the CCA se-

quences at the 3’ termini of substrates Ml RNA.

for cleavage

by

Results Cleavage of pTyr by Ml RNA from E. coli Ml RNA from E. coli can cleave pTyr (precursor to E. coli tRNATyr) su3+ in vitro in buffers that contain 60 mM MgClp or IO mM MgC12 plus 5 mM spermidine or spermine (Guerrier-Takada et al., 1983). If pTyr is first digested with snake venom phosphodiesterase to remove four to five nucleotides from its 3’ terminus and then used as a substrate, the rate of cleavage by Ml RNA decreases approximately IO-fold, as shown in Figure 1A (compare lane 6 with lane 8). Lanes l-6 in Figure 1A illustrate the cleavage by Ml RNA of pTyr molecules that lack the CCA sequence. No appreciable cleavage of the modified substrates is apparent until 180 ng of Ml are included in the reaction mixture (lane 6). However, if untreated pTyr is used as substrate, approximately the same rate of cleavage as seen in lane 6 is achieved with just 18 ng of Ml RNA in the reaction mixture (lane 8) with correspondingly higher rates apparent as more Ml RNA is used (lanes 912). This difference in rates with the two pTyr substrates is not apparent when the reconstituted RNAase P complex (Ml RNA plus C5 protein; Kole and Altman, 1979; GuerrierTakada et al., 1983) is used as the source of catalytic activity, as shown in Figure IB. We reported previously that C5 protein enhances the reaction rate when combined with Ml RNA (Guerrier-Takada et al., 1983; Guerrier-Takada and Altman, 1984) but does so to the same extent when either treated or untreated pTyr is used as substrate (compare lanes 1-6 with lanes 7-12). Furthermore, the rates are virtually identical when the same amount of Ml RNA (in the complex) is in the reaction mixture. When the reaction is carried out by Ml RNA alone in buffer that contains IO mM MgCI, and 5 mM spermine, the sensitivity to the presence or absence of the CCA sequence is still observed (data not shown). That the CCA sequence is indeed missing from the treated pTyr molecules is apparent from the somewhat faster mobility of this substrate in comparison to untreated pTyr as seen in Figure 1 and the analysis shown in Figure 2. pTyr treated with snake venom phosphodiesterase was digested to completion with RNAase T, and the products of digestion were separated on a 25% polyacrylamide sequencing gel (Figure 2, lanes 2-5: the limited nature of the digest is obvious in the figure). The largest oligonucleotide produced by RNAase T, digestion of pTyr contains 22 nucleotides proximal to the 3’ terminus and is marked in the figure, as are some of the other products. A calibration of the mobilities of the oligonucleotides shown in Figure 2 indicates that five nucleotides have been removed from the 3’ terminus of the treated molecule. The absence of CCA was confirmed by examination of the stoichiometry of the mononucleotide products resulting from phosphodiesterase digestion of pTyr (data not shown). The action of the exonuclease stops at or near the beginning of the

Cdl 220

(A) Cleavage by Ml RNA of intact and venom phosphodiesterase-treated precursor to E. coli tRNATY. Assays for the Ml RNA-catalyzed reaction were carried out rn buffer C (50 mM Tris-HCI, pH 7.5, 100 mM NH&I, 60 mM MgCI?, 5% glycerol) at 37°C for 15 min using pTyr-CA, defined as pTyr from which CCA has been removed, (lanes l-6), and intact pTyr (lanes 7-12) as substrates with various amounts of Ml RNA added as indicated below. The reaction products were separated on a 10% nondenaturing polyacryfamide gel at room temperature (Guerrier-Takada et al., 1983). The substrates are marked as ptRNA in the figure, while the 3’.proxrmal fragments of cleavage that contain the mature tRNA sequence are marked as tRNA and the 5’.proximal fragments as 5’. Lanes 1 and 7: no Ml RNA added; lanes 2 and 8: 18 ng Ml RNA added; lanes 3 and 9: 36 ng Ml RNA added; lanes 4 and 10: 60 ng Ml RNA added; lanes 5 and 11: 120 ng Ml RNA added: lanes 6 and 12: 180 ng Ml RNA added. (6) Cleavage by reconstituted RNAase P of intact and venom phosphodtesterase-treated precursor to E. co11 tRNATY. RNAase P was reconstituted by mixing Ml RNA (18 ng) and C5 protein (2-10 ng) in 120 pl buffer 6 (50 mM Tris-HCI, pH 7.5, 66 mM NH&I, 10 mM MgCI,) that contained pTyr-CA (lanes l-6) or Intact pTyr (lanes 7-12). Incubation was carried out at 37OC and 20 pl aliquots were withdrawn at the following times: 0 min (lanes 1 and 7); 2 mm (lanes 2 and 8): 5 min (lanes 3 and 9): 8 min (lanes 4 and IO): 12 min (lanes 5 and 11); 15 min (lanes 6 and 12). The reactions were then stopped and analyzed rn a 10% polyacrylamide gel as indicated‘in the legend to (A).

base-paired stem of the tRNA moiety of pTyr (Figure 3) but not before the terminal CA of the CCA sequence has been removed. B. C. Stark (Ph.D. thesis, Yale University, 1977) and Altman et al. (1974) observed an effect of the removal of 3’terminal nucleotides on the action of partially purified RNAase P, which depended on the amount of carrier tRNA in the reaction mixture. We therefore decided to investigate further the ability of bulk tRNA with and without CCA to inhibit the cleavage of pTyr by Ml RNA. As shown in Figure 4, lanes 1-4, bulk tRNA is an efficient inhibitor of this reaction. However, if the bulk tRNA itself is treated with snake venom phosphodiesterase to remove 3’.terminal nucleotides, it functions poorly as an inhibitor of the reaction (Figure 4, lanes 5-7). Indeed, even when as much as 5 pg of bulk tRNA from which CCA has been removed is included in the reaction mixture, no inhibition is apparent. In separate experiments we have shown that the reconstituted RNAase P complex (Ml RNA plus C5 protein) is inhibited to the same extent by bulk tRNA whether or not it had been treated with snake venom phosphodiesterase. The same results are observed with reconstituted RNAase P, regardless of the presence or absence of CCA in the pTyr substrate (data not shown). All these data are consistent with the possibility that the CCA sequence in the substrate influences the ability of Ml RNA, but not of reconstituted RNAase P, to bind to the tRNA moiety of tRNA precursor molecules. Accordingly, we measured the K, of the reaction with pTyr from which CCA had been removed, since, as we previously reported, Ml RNA alone appears to be responsible for binding of substrate. The results of this experiment showed that the K, for the substrate without CCA was 2.5 X 10d7 M-‘, a value within experimental error of that determined previously for the K, with the intact substrate, 4 x 10e7 M-’ (Guerrier-Takada

et al., 1983). We note, however, that the turnover number of the reaction with Ml RNA and pTyr without CCA is 0.05 mole/min/mole enzyme whereas the corresponding number with intact pTyr is 1.5 mole/min/mole enzyme. Preliminary studies indicate that the mode of inhibition by bulk tRNA is noncompetitive. Cleavage of a Human tRNA Precursor Molecule Eucaryotic tRNA genes do not encode the CCA sequence at the 3’ termini of mature tRNAs, and consequently, eucaryotic tRNA precursor molecules do not contain this sequence (see Altman et al., 1982). To explore further the reaction of Ml RNA with tRNA precursors that lack CCA, we used as substrate the precursor to human tRNAFMe’ transcribed in vitro (pFMet-precursor to human tRNAFMe’). This tRNA precursor molecule has eight extra nucleotides at its 5’ terminus and eight extra nucleotides at its 3’ terminus (Zasloff et al., 1982). As shown in Figure 5, lane 2, under the conditions we used, Ml RNA does not cleave this substrate. When the reaction is carried out with the reconstituted complex of RNAase P or crude RNAase P, the results are different. When a substrate containing the CCA sequence (pTyr) is used, the C5 protein cofactor does enhance the rate of cleavage by a factor of four under optimal conditions (unpublished experiments) compared to the rate of cleavage with Ml RNA alone. As is apparent in a comparison of Figure 5, lane 3, the “stimulation” of cleavage of the human precursor tRNA substrate is very great since there is no apparent rate of cleavage with Ml RNA (18 ng) alone. In this case it appears that the presence of C5 protein alters the ability of Ml RNA to interact with the substrate to such an extent that to speak of the stimulation of the reaction rate is misleading. However, we have observed that pFMet is cleaved in a reaction mixture containing a 20-fold higher concentration of Ml

Importance 221

of CCA rn Ml RNA Substrates

AU G A

3’-

q$on ;.5

6:; ::; E.2

$-CA-

,,,G-CAGGCCAGUAAAAAGCAUUACCCCG.C

ace-

G.C 5::: G~CCUUC%~~ . . .. .

U ~A&eouuC,, GCEAGcccU Uu.5 . .. C,“,A AAG$$GAG . CC .cA c. &,’ .G~

TUCG-

G

3OA.U G.C A.U*Q C A U A rsCUA

Figure 3. The Nucleotide Sequence and Presumed the Precursor to E. coli tRNATY su3+

G

Figure 2. Identification of Oligonucleotides phodiesterase Treatment of pTyr

Altered by Snake Venom Phos

pTyr treated with venom phosphcdiesterase as described in the Experimental Procedures was digested with RNAase Tl (5 ~1 of 5OC0 U/ml for 30 mm at 37OC) and the resulting mixture of oligonucleotides was electrophoresed in a 25% polyacrylamide sequencing gel. Lanes 1 and 6: untreated pTyr; lanes 2-5: pTyr digested with phosphodiesterase for 5, IO. 30, and 60 min. The largest oligonucleotide, 22 nucleotides long, contained 19 nucleotides proximal to the 3’ terminus of tRNA”’ and three extra nucleotrdes (Goodman et al., 1970; Schedl et al., 1974). It is marked as 3’ in the hgure. whereas the equivalent oligonucleotide from treated pTyr is marked as 3’CA. All other oligonucleotides are similar in the treated and control samples. Note the fragment that combines the anticodon (12 nucleotrdes long), which is marked as ace and appears as a doublet because of a mixture of molecules with different extents of nucleotide modification; the tetranucleotide, TWZG; and the mononucleotide. G.

RNA. When the reaction is carried out in buffer containing IO mM MgC12 and 5 mM spermine, no cleavage of substrate is observed (Figure 5, lane 4). We do not know, of course, if the equivalent subunits of RNAase P from human cells would behave in a similar fashion to the E. coli RNAase P subunits with the human precursor tRNA as substrate. If human RNAase P exhibits the same specificity as the enzyme from E. coli, the 3’ CCA sequence must be generated prior to tRNA precursor cleavage in human cells.

Cleavage of Bacteriophage T4 tRNA Precursor Molecules The pathway in vivo of the biosynthesis of six different T4 tRNAs (which code for proline, serine, threonine, isoleutine, glutamine, and leucine) from three different dimeric precursor molecules (Pro-Ser, Thr-lie, and Gin-Leu) is complex (Guthrie, 1975; Guthrie and Scholla, 1980; Seidman and McClain, 1975; Seidman et al., 1975; McClain, 1977). Only the Gln-Leu precursor contains the 3’ CCA after transcription (Fukada and Abelson, 1980). However, all three must terminate with 3’ CCA for RNAase P to cleave the central region of the dimeric precursors. Furthermore, the tRNAs that are encoded without the CCA sequence found in mature tRNAs must have their 3’-proximal extra

CAy,

Secondary

Structure

of

The sketch shows pTyr wrth the tRNA morety In the clover-leaf configuration and the nucleotrdes in the mature tRNA numbered. No nucleotrde modificatrons are shown though normally A36 + isA plus ms2iBA, U40 + Jr, U63 -+ T, U64 + Y. etc. The arrow at posrtron 1 of the mature tRNA sequence IS the sate of cleavage by RNAase P. The boxed nucleotides are not part of the 3’ terminus of the mature tRNA. Note the two hydrogen bonds formed between nucleotides -1 and 82, and -2 and 83.

PWC

TYI-

5,.

Figure 4. Inhibition of the Reaction Catalyzed by Ml RNA by Intact tRNA and tRNA Treated wrth Venom Phosphodiesterase pTyr was incubated wrth Ml RNA (18 ng) in buffer C for 15 min at 37°C in the absence (lane 9) or presence of bulk tRNA from E. coli (lanes l-4: 0.1, 0.5, I, and 2 pg. respectively) or bulk tRNA from which the terminal CCA has been removed as described in Experimental Procedures (lanes 5-7: 0.1, 0.2, 0.5 pg, respectively). Five micrograms of treated tRNA also has no effect on the reaction: data not shown. Lane 8: pTyr incubated without Ml RNA. Lanes 8 and 9 were run on a separate gel and the products, whrch correspond to those seen in lanes l-7, have moved further down. Tyr refers to the 3’ fragment of Ml RNA cleavage of pTyr. which contains the mature tRNA sequence, and 5’ is the cleavage product containing the extra 5’.proximal nucleotides.

nucleotides removed by an exonuclease and the CCA sequence added by nucleotidyltransferase prior to cleavage by RNAase P at the 5’ terminus of the mature tRNA sequence. This requirement for processing of the 3’ terminus prior to 5’ endonucleolytic cleavage is not readily reproduced in vitro with crude RNAase P preparations

Cell 222

Frgure 6. Cleavage of Bacteriophage T4 Pm-8er cursor tRNA Molecules by RNAase P

1-w

Figure 5. Cleavage

by RNAase

P of a Precursor

to Human tRNkM

pFMet transcribed in vitro (see Experimental Procedures) was used as substrate for Ml RNA (18 ng) or reconstituted RNAase P (18 ng Ml RNA plus 2-10 ng C5 protein) as indicated betow. After incubation at 37°C for 15 min. the products were analyzed in a 5% polyacrylamide gel containing 7 M urea, 50 mM Tris-borate, pH 8.3, 1 mM EDTA. Lane 1: pFMet only; lane 2: pFMet plus Ml RNA in buffer C; lane 3: pFMet plus Ml RNA reconstituted with C5 protein in buffer B; lane 4: pFMet plus Ml RNA plus 5 mM spermine in buffer B; lane 5: pFMet plus C5 protein (2-10 ng) in buffer B; lane 6: pTyr alone; lane 7: Ml RNA plus pTyr in buffer C. The oligonucleotide released from the 5’ end of pFMet by RNAase P cleavage has run off the bottom of the gel.

(Schmidt and McClain, 1978; Guthrie and Scholla, 1980). We have examined the action in vitro of Ml RNA on the three dimeric precursors. Ml RNA exhibits the same specificity for cleavage in vitro as has been described for the T4 tRNA biosynthetic pathway in vivo. Note in Figure 6 that with the Pro-Ser (lane 2) and Thr-lie precursor (lane 7) molecules are cleaved at low rates by Ml RNA alone as compared with the Gln-Leu precursor, which is cleaved readily at the 5’ terminus of the Leu sequence (Figure 7, lanes 2-5). However, cleavage at the 5’ terminus of the monomeric Gln precursor, which lacks CCA (product 1 in Figure 7) is also much slower than cleavage at the 5’ terminus of the Leu sequence in the dimeric precursor. All cleavage products were fingerprinted to ascertain that cleavage by Ml RNA occurred at position 1 of each mature tRNA sequence (see Seidman and McClain, 1975; Seidman et al., 1975; Guthrie and Scholla, 1980, for details). The cleavage products corresponding to mature tRNAn’ and tRNAle are similar in size and are not resolved in the electrophoretic system we used. However, when reconstituted RNAase P or crude RNAase P is used as the source of catalytic activity, the sensitivity to the presence or absence of the CCA sequence in the substrates is not apparent. That is, the rate of reaction with the Pro-Ser and Thr-lie precursors (Figure 6, lanes 4 and 5) is enhanced at least 5-fold (as determined by visual inspection) by the presence of C5 protein. If a Pro-Ser precursor is used that has already been partially processed in vivo so that about

and Thr-lie Dimeric Pre-

These precursors were prepared in E. coli BN and therefore have no 3’ CCA sequences. The dimeric tRNA precursors for Pro&r (lanes l-4) and Thr-lie (lanes 5-8) tRNAs were incubated at 37°C for 1 hr in the presence of Ml RNA (18 ng) plus 2-10 ng C5 protein as indicated below. The reaction mixtures were analyzed on a 10% nondenaturing polyacrylamide gel. Lanes 1 and 8: dimertc ptRNAs only; lanes 2 and 7: dimeric ptRNAs plus Ml RNA in buffer C; lanes 3 and 6; dimeric ptRNAs plus Ml RNA plus 5 mM spemrine in buffer 8; lanes 4 and 5: dimeric ptRNAs plus Ml RNA reconstituted with C5 protein in buffer B. Note that the products of cleavage of the Thr-lie precursor that contain the tRNA sequences are not resolvable rn this gel system. The Y-proximal fragments have run off the bottom of the gel. Lane 9: pTyr plus Ml RNA (18 ng) rn buffer C; lane 10: pTyr alone.

50% of the preparation contains CCA at the 3’ terminus, the rate of cleavage in vitro by Ml RNA increases accordingly (data not shown). The reaction rate at the 5’ terminus of the tRNALe” sequence in the Gin-Leu precursor is unchanged (Figure 7, lanes 6-9; see Figure 7, bottom, for an explanation of the products generated and shown in the top of Figure 7) when reconstituted RNAase P is used compared to that with Ml RNA alone. However, cleavage of the dimeric precursor by reconstituted RNAase P at the 5’ terminus of the tRNAG’” sequence is markedly enhanced. When the reactions are carried out with Ml RNA alone in the presence of spermidine, the sensitivity to the presence or absence of the CCA sequence is apparent (Figure 6, lanes 3 and 6; Figure 7, lanes 10-13). Note the low rate of production of product 3 from product 1 (products defined in the legend to Figure 7) in the presence of spermine in comparison with the rate observed when the reconstituted enzyme is used as shown in lanes 6-9. Finally, if the GlnLeu precursor is treated with venom phosphodiesterase, the rate of cleavage of the treated substrates by Ml RNA decreases markedly in comparison to that observed with intact precursor (data not shown). The ability of C5 protein in the reconstituted RNAase P complex to diminish substantially the sensitivity of the enzyme to the presence of the CCA sequence in its substrates is also apparent from an examination of the reactions shown in Figure 6, lane 4 and Figure 7, lanes 69. In contrast to the reactions seen with Ml RNA alone, using the T4 substrates, when the reconstituted RNAase P is used as enzyme, a doublet band appears clearly in

Importance 223

of CCA in Ml RNA Substrates

pAAUAAU&i&mCCA 4 RNasc r.mCCA

1. pAAUAAUmC 4 RNase

P

P

diiilC Figure 7. Cleavage of Bacteriophage Molecule by RNAase P

T4 Gin-Leu Dimeric tRNA Precursor

Thus precursor has the 3’ CCA sequence, whether prepared in E. coli A49 (as this was) or BN. The Gin-Leu dimeric tRNA precursor was incubated at 37°C in the presence of 18 ng Ml RNA (lanes 2-5: 2, 5, 10, 15 min, respectively) in buffer C, the reconstituted RNAase P complex in buffer B (18 ng Ml RNA plus 2-10 ng C5 protein; lanes 6-9: 2. 5. 10, 15 min, respectively) or 18 ng Ml RNA plus 5 mM spermine in buffer B (lanes IO13: 2. 5, 10. 15 min, respectively); lane 1: Gln-Leu dimerii precursor only; lane 14: Gln-Leu dimeric precursor incubated with C5 protein in buffer B. The substrate is denoted as K in the upper part of the figure (Guthrie. 1975). The expected order of cleavages by RNAase P and the resultant products are shown and numbered in the bottom part of the figure.

the ptRNA position after 15 min of incubation of the reaction mixture. The faster-migrating species results from cleavage of the intact dimeric precursors at position 1 of the 5’-proximal tRNA sequence, which releases, as a consequence, a small oligonucleotide by comparison to the total size of the substrate molecule. While this cleavage site seems more accessible to intact RNAase P than to Ml RNA, the results show that the first cleavage occurs predominantly at position 1 of the 3’-proximal tRNA sequence. The data collected from experiments with T4 tRNA precursors reflect exactly the results seen with the monomeric precursor molecules that were discussed above. We have also observed in separate experiments (data not shown) that pM1 RNA, the precursor to Ml RNA (GuerrierTakada and Altman, 1984) can carry out the same reactions as Ml RNA with the T4 tRNA precursor molecules. Discussion

We have shown that Ml RNA from E. coli, an RNA molecule that possesses catalytic activity, is sensitive to the presence or absence of the CCA sequence at the 3’

termini in its tRNA precursor substrate molecules by observation of the rates of reaction with various substrates. In particular, by treating pTyr with snake venom phosphodiesterase to remove 3’ nucleotides, we created an altered substrate that was much less susceptible to cleavage by Ml RNA than the untreated substrate. Similar low rates of cleavage of the precursor to human tRNAFMe’ and certain T4 tRNA precursors that lack CCA (Pro-Ser and Thr-lie precursors) have also been observed. By contrast, a T4 tRNA precursor that had the CCA sequence (Gin-Leu precursor) was cleaved at a relatively high rate by Ml RNA in vitro. Furthermore, bulk E. coli tRNA, normally an efficient inhibitor of the Ml RNA-catalyzed reaction, no longer had this property when CCA sequence was removed by treatment with exonuclease. The K, of the Ml RNA reaction with the pTyr substrate is unchanged but the turnover number is greatly reduced when the 3’terminal nucleotides, including the CCA sequence, are absent. These data are consistent with the hypothesis that one aspect of the Ml RNA catalytic process is the formation of bonds with invariant nucleotides in the tRNA moieties of tRNA precursor molecules (Reed et al., 1982). Attention was focused previously on interactions during substrate recognition with the GTQCG sequence in the tRNA moieties because of a complementary pentanucleotide sequence in Ml RNA. In light of the present results, we now recognize five trinucleotides in Ml RNA complementary to CCA, but no direct test has been made of the interaction of these sequences with CCA in the substrates. Since Ml RNA is encoded by the E. coli chromosome and all tRNA genes in E. coli encode CCA sequences, there may be a speciesspecific interaction between these sets of molecules that is not seen in cross-species experiments. It may be that in human cells the RNA subunit of RNAase P is efficient in recognizing its homologous substrates or, as we observed for E. coli RNAase P, the corresponding protein cofactor alters the specificity of the enzyme. Indeed, we have shown here that the presence or absence of the CCA sequence in substrate molecules is immaterial when the reconstituted RNAase P complex, containing protein and RNA, is used as the source of catalytic activity in vitro. Spermine or spermidine cannot substitute for the protein cofactor in this recognition process. Thus the protein, as we observed previously (Guerrier-Takada et al., 1983) is not acting merely as a basic counterion in vitro. The significance of our observations to the physiology of T4 infection may be considerable. Certain T4 tRNA precursors containing the 3’ CCA sequence are processed in vivo by RNAase P more readily than those not having this sequence. Similarly, we have shown that Ml RNA in vitro cleaves tRNA precursor sequences with 3’ CCA much more readily than those lacking this sequence. When reconstituted RNAase P is used to carry out the cleavage reaction in vitro, these dramatic differences in rates are not seen. In vivo, not only is the order of RNAase P action on T4 tRNA precursors specified in a unique way in comparison with its action on E. coli tRNA precursors, but also the half-lives of those T4 tRNA precursors that

Cell 224

lack 3’ CCA are 50-l 00 times longer than the half-lives of the precursors that contain 3’ CCA and at least one E. coli substrate, pTyr (Altman, 1971; Guthrie et al., 1973). Seidman et al. (1975) observed RNAase P cleavage in vivo of a bacteriophage T2-encoded Pro precursor that lacked the 3’ CCA. They note the possibility that the phage may modify host-processing enzymes to achieve certain phage-specific functions. Indeed, the exact nature of RNAase P specificity in vivo may depend strongly on whether and how the activity of the enzyme is modified during phage infection. Experimental

Received

Chemicals and Enzymes All chemicals were reagent grade. Acrylamtde and bis-acrylamide were purchased from Brorad Corp. Ultra-pure urea was obtained from SchwarzMann. Venom phosphodiesterase from Vrpera lebetina was purchased from Calbrochem-Boehringer. Radioactrve materials were purchased from Amersham Radiochemrcals Corporation. Preparation of Ml RNA and CS Protein Ml RNA was prepared as described by Read et al. (1982). C5 protein was prepared as described by Guerrier-Takada et al. (1983). Preparation of Radioactive Substrates for RNAase P Preparatron of pTyr was carried out as described by Robertson et al. (1972). Bacteriophage T4 tRNA precursor molecules were prepared according to Guthrie et al. (1973) Serdman and McClarn (1975) and Seidman et al. (1975). Precursor to human tRNArm transcribed rn vitro was a generous gaff of J. Tobran (NIH; Zasloff et al., 1982). Partial Digestion of RNA with Snake Venom Phosphodiesterase pTyr as Substrate pTyr (5 x 105 cpm) was suspended in 125 pl phosphodiesterase buffer (50 mM Tns-HCI, pH 8.9, IO mM MgCb). Twenty-five microliters were withdrawn for the control “minus enzyme.” To the remaining 100 ~1 we added 1 pl venom phosphodiesterase (6.5 X 10m3 U) and the mixture was Incubated at room temperature. Aliquots of 25 ~1 were withdrawn at 5, 10, 30, and 60 min. Each of these akquots and the control sample was added to 5 pl liquefied phenol to Inactivate the enzyme. The RNA was then precipitated with ethanol in the presence of carrier tRNA. The radioactive products of digestion were separated on a 5% polyacrylamide gel (containing 7 M urea, 50 mM Tns-borate. pH 8.3, 1 mM EDTA) and were visualized by autoradiography. Slices at the positions of intact pTyr and phosphodiesterase-treated pTyr were excused from the gel and eluted by crushing and soaking in TE buffer (IO mM Tns-HCI, pH 7.5, 1 mM EDTA). Elution was carried out for several hours at 4’C. The eluted RNA was lyophilized, washed with 75% ethanol, assayed for radioactivity, and then used as substrate for Ml RNA and the reconstrtuted RNAase P complex. Bulk tl?NA as Substrate Bulk E. co11tRNA (140 Gg) was suspended in 100 pl of phosphodiesterase buffer and drgested with 1.3 X IO-’ U of venom phosphodresterase for 30 mm at room temperature. The reaction was stopped with 5 pl lrquefied phenol and the RNA was precipitated with ethanol. A parallel sample was treated as a control rn an identical fashion except that no enzyme was added to the mixture. The RNA pellets were washed twice with 75% ethanol, lyophrkzed. suspended in water, and then tested as inhibrtors of the RNAase P reaction. Assays for RNAase P Acttvity These were carried out as described prevrously (Guerner-Takada et al., 1983). Condrtions were adjusted so that most assays reported here were carried out rn the linear portion of the curve describing kinetrcs of cleavage.

for technical

assrstance

and to Dr.

supported

by

in part by the hereby marked 1734 solely to

June 6, 1984; revised

References Akaboshi, E., Guerrier-Takada, C., and Altman, S. (1980). Veal heart RNAase P has an essential RNA component. Biochem. Brophys. Res. Commun. 96, 831-837. Altman, S. (1971). Isolation of tryosine New Biol. 229, 19-21.

Procedures

We are grateful to Donna Wesolowski

Ann Korner for help with the manuscript. This work was grants from the NSF and USPHS to S. A. and to W. H. M The costs of publication of this artrcle were defrayed payment of page charges. This article must therefore be “advertisement” in accordance with 18 U.S.C. Section indicate this fact.

tRNA precursor

molecules.

Altman, S., Bothwell, A. L. M., and Stark, B. C. (1974). Processing co11 tRNATv precursor tn vitro. Brookhaven Symp. Biol. 26, 12-25.

Nature of E.

Altman, S., Guerrier-Takada, C., Frankfort, H., and Robertson, H. D. (1982). RNA processing nucleases. In Nucleases, S. Linn and R. Roberts, eds. (Cold Spnng Harbor, New York: Cold Spring Harbor Laboratory). Fukada. K., and Abelson, J. (1980). DNA sequence gene cluster. J. Mol. Biol. 139. 377-391.

of a T4 transfer

RNA

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of a dimeric

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