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Kinetics of the processing of the precursor to 4·5 S RNA, a naturally occurring substrate for RNase P from Escherichia coli
J. MoZ. Biol. (1991) 221, 1-5
COMMUNICATIONS
Kinetics of the Prkessing of the Precursor to 4-5 S RNA, a Naturally Occurring Substrate for RNase P from Escherichia coli Karen A. Peck-Millert
and Sidney Altman
Department of Biology, Yale University New Haven, CT 06520, U.S.A. (Received
10 December
1990; accepted 25 April
1991)
A study was made of the cleavage by Ml RNA and RNase P of a non-tRNA precursor that can serve as a substrate for RNase P from Escherichia co&, namely, the precursor to 45 S RNA (~45s). The overall efficiency of cleavage of p45S by RNase P is similar to that of wild-type tRNA precursors. However, unlike the reaction with wild-type tRNA precursors, the reaction catalyzed by the holoenzyme with p45S as sub&r&e has a much lower K,,, value than that catalyzed by Ml RNA with the same substrate, indicating that the protein subunit plays a crucial role in the recognition of ~4.5s. A model hairpin substrate, based on the sequence of p4*5S, is cleaved with greater eficiency than the parent molecule. The 3’-terminal CCC sequence of ~45 S may be as important for cleavage of this substrate as the 3’-terminal CCA sequence is for cleavage of tRNA precursors. Keywords: RNase P; 4.5 S RNA precursor; enzyme kinetics; Ml RNA
RNase P from Escherichia wli is capable of cleaving a variety of molecules in vitro that are not precursors to tRNAs, the natural substrates of this enzyme in vivo. These molecules include fragments of the 3’ terminus of turnip yellow mosaic virus (TYMV; Guerrier-Takada et al., 1988; Green et al., 1988); M, RNA, encoded by bacteriophage 480 (Bothwell et al., 1976a); small hairpin substrates with sequences based on the sequence of various tRNAs (McClain et al., 1987; Forster & Altman, 1990; K. Peck-Miller & S. Altman, unpublished results), and the precursor to 4.5 S RNA (Bothwell et al., 1976b). It is clearly of some interest to elucidate the way in which RNase P can recognize all these different molecules as substrates. Unlike the other substrates that are not precursors to tRNAs, the precursor to 45 S RNA (p4*5S$) from E. coli is the only naturally occurring nontRNA precursor substrate for RNase P that is known to be processed both in vivo and in vitro. In addition, the secondary structure of p45S resembles a long hairpin (Hsu et al., 1984) and is quite different from the secondary structures of the tRNA
portions of precursor tRNAs, which resemble a cloverleaf. Mature 45 S RNA is a stable RNA that interacts with ribosomes during translation (Brown & Fournier, 1984; Bourgaize & Fournier, 1987; Brown, 1987) and which appears to play a role in secretion of certain proteins (Ribes et al., 1990). The study of p45S has now been simplified through the construction of a clone for use in preparation of this RNA in vitro. The kinetic parameters of the reaction with the RNase P holoenzyme (Ml RNA plus C5 protein), or with Ml RNA, its catalytic subunit, alone (Guerrier-Takada et al., 1983), and p45S RNA have been determined, and the effect of deletions in the hairpin structure or 3’terminus of the substrate have provided information on the relative importance of these features for recognition and efficient cleavage by the enzyme. The RNA substrate ~23-4.55, which is identical to wild-type p45S except for the deletion of a single C residue at the second position of the wild-type transcript, was transcribed in vitro from a linearized DNA template using phage T7 RNA polymerase (Fig. 1). Three derivatives of the substrate ~23-455 were also transcribed, each of which contained a stem of only 14 base-pairs in the sequence of the mature molecule instead of 42 base-pairs. These smaller derivatives were designated pS14-CCA, pS14-CCACCC and pS14-CCACCA (Fig. l), and they
7 Present address: Bristol-Meyers Squibb Pharmaceutical Research Institute, 3005 First Avenue, Seattle, WA 98121, U.S.A. $ Abbreviation used: p45S, precursor to 45 S RNA. 1 0022-2836/91/17000145
$03.00/O
0
1991 Academic Press Limited
K. A. Peck-Miller
2
and 8. Altman
~23-4.5s
pS14-CCA
pS14-CCACCC
pS14-CCACCA
5’pppGC_CGCUCUC~u_A~3 U-A G-C U-A g:: GC u uu c
G
G-C G-C C-G U-G C-G U-A G-C
;:g G-U G-C
,P
U G
CAU
Figure 1. Proposed secondary structures of derivatives of ~4.5s RNA. ~23-4.56 is identical to wild-type ~4.58 except for the deletion of a single C residue in the 2nd position of the wild-type transcript (Hsu et al., 1984). The boxed region of ~23-45s indicates nucleotides that were deleted to create pS14-CCACCC. pS14-CCA. pS14-CCACCA and pS14-CCACCC differ in sequence only at the 3’ terminus. The arrow indicates the site of cleavage by MI RNA or RNase P. Each RNA was prepared in vitro from a linearized DNA template using phage T7 RNA polymerase and then gel-purified, as described (Peck, 1990). Sites of cleavage were determined by end-group analysis and by calibration of cleavage products with fragments of known size in sequencing gels (Peck, 1990).
differ from one another with respect to the sequence at the 3’ terminus. The proposed secondary structure of the smaller substrates resembles the small tRNA-like hairpin substrates that are processed efficiently in vitro by RNase P from E. coli (McClain et al.,1987; Forster & Altman, 1990; K. Peck-Miller & S. Altman, unpublished results). Moreover, deletions were positioned such that the sequence GUUCA appeared in the loop region of the small ~23-4.5s derivatives at a position analogous to that of the GUUCG sequence in the small tRNA-like hairpin substrates. These sequences resemble the conserved GTYCR sequence in the T loop of tRNA precursors. A kinetic analysis was made by cleavage of ~23-4.5s and pS14-CCACCC (which have identical 3 termini) by using Ml RNA, the catalytic RNA subunit of RNase P, or RNase P as enzyme (Table 1). Since each kinetic parameter could be reproduced experimentally within a factor of about
2, a difference of threefold or more between kinetic values was considered to be significant. A comparison of the kinetics of cleavage of the precursor to a natural tRNA (pTyr) and a nontRNA (~23-45s) by RNase P from E. coli reveals that the overall efficiency of the reaction (k,,,/K,) with RNase P is similar for each substrate (the relative abundance in the cell of a single tRNA species and 4.5 S RNA is also similar). However, the overall efficiency of the cleavage reaction catalyzed by Ml RNA alone is 11 times greater for pTyr than for ~23-455, and this difference is mostly due to a difference in the K,,, value. For p23-4.5S, the K,,, value of the reaction is decreased Wfold in the presence of C5 protein, the protein subunit of RNase P (Table 1). The effect on the K,,, value is significantly different from that observed with wild-type tRNA precursors, in which case the K, of the reaction catalyzed by RNase P is similar to the K, of the reaction catalyzed by Ml RNA (McClain et al.,
Communications
Table 1 Kinetic parameters of cleavage of substrates derived from pd.53 RNA by Ml RNA and RNase P
( PTY~S p2345s ~814.CCACCC
Ml RNase P Ml RNase P Ml RNase P
XT& 006 903 17.0 62 25 004
&aJKm
lc,,,t
92 66 43 37.0 22.0 290
(x 10-s) 3.3 220.0 93 190.0 8.8 5000
Identical radioactively labeled ([a-32P]GTP) and non-radioactively labeled substrate RNAs were mixed and used for all assays (Peck, 1990). Time points at which the reactions were stopped were adjusted so that measurements were taken on the linear portion of the curve of kinetics of the cleavage reaction. Reactions with Ml RNA as enzyme were carried out in buffer A, and reactions with RNase P as enzyme were carried out in buffer B (described in the legend to Fig. 2). Concentrations of substrates used were optimal for Michaelis-Menten kinetics, and values of K, and V,,. were obtained from Hanes-Woolf plots (Fersht, 1985; Segel, 1975). Each kinetic parameter is the average of results from 2 to 4 independent experiments and each was reproducible within a factor of about 2. t k,,, = mol product/min per mol enzyme. f: From Guerrier-Takada et al. (1988).
1987; Reich et al., 1988; Guerrier-Takada et al., 1988). The distinctive interactions of the hairpin structure of 4.5 S RNA and the cloverleaf structures of tRNAs with the enzyme might explain the difference in the kinetic parameters of their cleavage reactions. Because the K,,, value for the reaction with Ml RNA and wild-type tRNA precursors does not change by more than 50% with the addition of C5 protein, it is likely that the Ml RNA molecule can achieve the correct, or nearly correct conformation for recognition of tRNA substrates without the aid of the protein cofactor. This capability appears to be compromised somewhat in the reaction with p23-45S, as evidenced by the high K,,, value for the reaction with Ml RNA: therefore, C5 protein plays a more significant role in aiding Ml RNA to achieve the necessary conformation of the enzymesubstrate complex with the non-tRNA substrate. Chemical modification of crosslinked complexes of Ml RNA and a tRNA precursor substrate revealed that the substrate may unfold during processing (Knap et al., 1990). Since longer helices are more stable than shorter ones (Freier et al., 1986), the helical structure of ~23-455 may be unfolded less readily than those of tRNA precursors, an effect that could increase the effective K, value of the reaction with ~23-4.5s if unfolding is part of the binding process. The presence of the very basic C5 protein may facilitate unfolding of ~23-4.55, thereby decreasing the effective K,,, value. In fact, in support of these assertions, we found that removal of a large portion of the hairpin of ~23-45s results in a substrate (pSl4-CCACCC) that has a higher k,,, and lower K,,, value for the reaction with Ml RNA compared with the parent substrate (Table 1). The
3
overall efficiency of processing of pSl4-CCACCC by Ml RNA is approximately 30-fold greater than that observed for ~23-4.55. Furthermore, the overall efficiency of cleavage of pSl4-CCACCC by RNase P is slightly greater than that observed for p23-4.5S, mostly as the result of a lower K,,, value for the reaction with pS14-CCACCC. C5 protein has a similar effect on the efficiency of cleavage of each substrate: the decrease in K,,, value of the reaction is much more dramatic than the increase in k,,, value of the reaction in the presence of C5 protein. The portion of the stem that was deleted from ~23-45s is not required for cleavage to take place. Moreover, since pSl4-CCACCC is cleaved at the expected site, it is clear that features of the substrate that are in close proximity to the cleavage site are sufficient for selection of the cleavage site, and that structural features further from the cleavage site are dispensable for this process. The increased overall efficiency of the processing of pSl4CCACCC, compared with that of p23-4.5S, may be due to the fact that pSl4-CCACCC resembles a tRNA precursor in terms of structure more closely than ~23-4.5s does. The helix of 14 base-pairs in the mature portion of pSl4-CCACCC is similar to the helical region of 12 base-pairs near the site of cleavage of tRNA precursors, which results from stacking of the acceptor stem and the T stem (McClain et al., 1987). Since the invariant 3’-terminal CCA sequence of both precursor tRNAs and small tRNA-like precursors is important for cleavage by RNase P from E. coli (Guerrier-Takada et al., 1984, 1989; McClain et al., 1987; K. Peck-Miller & S. Altman, unpublished results), an analysis was made of the importance of the 3’-terminal nucleotides of the substrate pSl4CCACCC. Variants of pSl4-CCACCC were tested that terminated in either a single-stranded 3’ CCA (pSl4-CCACCA) or a double-stranded region containing the “internal” 3’ CCA (pSl4-CCA; Fig. I), instead of the wild-type terminal 3’ CCC sequence. To our surprise, the substrates terminating in CCA were not processed as readily by the enzyme as the substrate that terminated in the wild-type CCC sequence (Fig. 2). Under the conditions used, no cleavage was detected by Ml RNA of pSl4-CCA, although limited cleavage by RNase P was apparent. In the case of pSl4-CCACCA, the extent of cleavage both by Ml RNA and by RNase P was greater than for pSl4-CCA, but the rate of cleavage was still below that of pSl4-CCACCC. Cleavage by Ml RNA is more sensitive to the nature of the 3’-terminal nucleotides of these substrates than is cleavage by RNase P, since the rate of cleavage by RNase P is not as severely affected by changes at the 3’ terminus. A similar pattern was observed with both minimal and full-length tRNA precursor substrates in the presence of C5 protein (Guerrier-Takada et aZ.,1984; Forster & Altman, 1990; K. Peck-Miller & S. Altman, unpublished results). Our results indicate that the 3’.terminal sequence of minimal substrates that resemble ~4.5s RNA is
4
K. A. Peck-Miller
and S. A&man
recursor
3’ Fr,agment -
5’ FIpogment
Figure 2. Cleavage by Ml RNA and RNase P of minimal p45S-like substrates. All substrates were transcribed in vitro in the presence of [c+~‘P]GTP, as described (Peck, 1990). Lanes 1 to 3, pS14-CCA with no enzyme added (lane l), with Ml RNA (lane 2), and with RNase P (lane 3). Lanes 4 to 6 pS14-CCACCC with no enzyme added (lane 4), with Ml RNA (lane 5), and with RNase P (lane 6). Lanes 7 to 9, pS1CCCACCA with no enzyme added (lane 7), with Ml RNA (lane 8), and with RNase P (lane 9). Reactions with Ml RNA were carried out in buffer A (50 mm-Tris. HCl (pH 7.5), 100 mM-NH,Cl, 500 mM-MgCl,) in the presence of 25 ng of Ml RNA for 14 min. Reactions with RNase P were carried out in buffer B (50 mM-Tris . HCl (pH 75), 100 mM-NH&I, 10 mM-MgCl,) in the presence of 3 ng of Ml RNA plus a lo-fold molar excess of C5 for 4 min. All reactions were carried out in a reaction volume of 10 ~1 at 37 “C. The products of the reactions were precipitated with ethanol (in the presence of 20 pg glycogen/ml as carrier) before electrophoresis on a 12% (w/v) acrylamide/7 M-urea gel, then they were visualized by autoradiography. Preparation of Ml RNA has been described (Vioque et al., 1988), and C5 protein was a generous gift from Dr A. Vioque.
important for the cleavage reaction, as it is for the cleavage of tRNA precursors and the minimal substrates derived from them. Despite the fact that a 3’-terminal CCA sequence is present in pS14-CCA, it is a poor substrate (Pig. 2). Therefore, the mere presence of CCA at the 3’ terminus is not enough to promote efficient cleavage of this RNA. The addition of a feature common to tRNA precursors, namely a single-stranded 3’.terminal CCA, to the minimal p4.5S-like substrate pS14-CCA to create pSl4-CCACCA does enhance the rate of cleavage, but not to the same rate as that observed with the analogous substrate that contains the wild-type 3’ terminus (pS14-CCACCC). From these experiments it is clear that some intrinsic feature of the 3’terminal nucleotides of pS14-CCACCC is important for cleavage of this substrate and, presumably, for cleavage of the precursor to 45 S RNA. Additional nucleotides beyond the internal CCA sequence in the double-stranded region are required for efficient cleavage, since cleavage of pS14-CCACCC and pS14CCACCA proceeds with greater efficiency than that of pS14-CCA (Fig. 2). The 3’-terminal CCA of all tRNA precursors is the specific CCA sequence that is important for the cleavage reaction, and the additional internal copies that exist in some substrates are not involved (Sprinzl et al., 1987). Likewise, the internal CCA sequence of p4_5S, which is adjacent to the 3’-terminal CCC, is unlikely to be important for the cleavage by RNase P. Guerrier-Takada et al. (1989) have indicated that the S/-terminal CCA of tRNA substrates interacts with a particular binding site on Ml RNA, although
the absence of the terminal A does not affect the overall rate of reaction (our unpublished results). This proposal may also be relevant to the role of the 3’-terminal CCC of ~4.5s. The interaction with the 3’ end of the substrate, in concert with other features of the substrate, may help to orient the enzyme for correct binding and cleavage of the substrate. This hypothesis is supported by the fact that cleavage of pre-tRNAs (with the exception of pHis) and ~4.5s always occurs in the same position relative to the terminal single-stranded CCA or CCC sequence (Fig. 3).
C C C
A C C
NC -NA
-NB
El .. tf?NA
NC -NA
-NB
El .. 4.5 s
Figure 3. Position of 3’-terminal nucleotides relative to the sites of cleavage of precursor tRNAs and fl5S RNA. Only the nucleotides near the sites of cleavage (arrow) are shown. N, nucleotide; and N, and N, are usually basepaired in tRNA precursors.
Communications We thank the members of our laboratory for helpful discussions. This work was supported by grants from the USPHS (GM19422) and USNSF (DMB8722644) to S.A. References Bothwell, A. L. M., Stark, B. C. & Altman, S. (1976u). PTOC.Nat. Acad. Sci., U.S.A. 73, 1912-1916. Bothwell, A. L. M., Garber, R. L. & Altman, S. (19765). J. Biol. Chem. 251, 7709-7716. Bourgaize, D. B. & Fournier, M. J. (1987). Nature (London), 325, 281-284. Brown, S. (1987). Cell, 49, 825833. Brown, S. & Fournier, M. J. (1984). J. MOE. Biol. 178, 533-550. Fersht, A. (1985). Enzyme Structure and Mechanism, W. H. Freeman and Company, New York. Forster, A. & Altman, S. (1990). Science, 249, 783-786. Freier, S. M., Kierzek, R., Jaeger, J. A., Sugimoto, N., Caruthers, M. H., Neilson, T. & Turner, D. (1986). Proc. Nat. Acad. Sci., U.S.A. 83, 9373-9377. Green, C. J., Vold, B., March, M. D., Joshi, R. L. & Haenni, A.-L. (1988). J. Biol. Chem. 263, 11617-l 1620. Guerrier-Takada, C., Gardiner, K., Marsh, T., Pace, N. & Altman, S. (1983). Cell, 35, 849-857.
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Guerrier-Takada, C., M&lain, W. H. t Altman, S. (1984). Cell, 38, 219-224. Guerrier-Takada, C., van Belkum, A., Pleij, C. W. A. & Altman, S. (1988). Cell, 53, 267-272. Guerrier-Takada, C., Lumelsky, N. & Altman, S. (1989). Science, 246, 1578-1584. Hsu, L. M., Zagorski, J. & Fournier, M. J. (1984). J. Mol. Biol. 178, 509531. Knap, A., Wesolowski, D. & Altman, 8. (1990). Biochimie, 72, 779-790. McClain, W. H., Guerrier-Takada, C. & Altman, S. (1987). Science, 238, 527-530. Peck, K. (1990). Ph.D. thesis, Yale University, New Haven, CT. Reich, C.,‘ Olsen, G. J., Pace, B. & Pace, N. (1988). Science, 239, 178-181. Ribes, V., Romisch, K., Giner, A., Dobberstein, B. & Tollervey, D. (1990). Cell, 63, 591-600. Segel, I. H. (1975). Enzyme Kinetics, John Wiley & Sons, New York. Sprinzl, M., Hartmann, T., Meissner, F., Moll, J. & Vorderwulbecke, T. (1987). NAR 15, r53-rl88. Vioque, A., Arnez, J. & Altman, S. (1988). J. Mol. Biol. 202, 835-848.
Edited by A. Klug
COMMUNICATIONS
Kinetics of the Prkessing of the Precursor to 4-5 S RNA, a Naturally Occurring Substrate for RNase P from Escherichia coli Karen A. Peck-Millert
and Sidney Altman
Department of Biology, Yale University New Haven, CT 06520, U.S.A. (Received
10 December
1990; accepted 25 April
1991)
A study was made of the cleavage by Ml RNA and RNase P of a non-tRNA precursor that can serve as a substrate for RNase P from Escherichia co&, namely, the precursor to 45 S RNA (~45s). The overall efficiency of cleavage of p45S by RNase P is similar to that of wild-type tRNA precursors. However, unlike the reaction with wild-type tRNA precursors, the reaction catalyzed by the holoenzyme with p45S as sub&r&e has a much lower K,,, value than that catalyzed by Ml RNA with the same substrate, indicating that the protein subunit plays a crucial role in the recognition of ~4.5s. A model hairpin substrate, based on the sequence of p4*5S, is cleaved with greater eficiency than the parent molecule. The 3’-terminal CCC sequence of ~45 S may be as important for cleavage of this substrate as the 3’-terminal CCA sequence is for cleavage of tRNA precursors. Keywords: RNase P; 4.5 S RNA precursor; enzyme kinetics; Ml RNA
RNase P from Escherichia wli is capable of cleaving a variety of molecules in vitro that are not precursors to tRNAs, the natural substrates of this enzyme in vivo. These molecules include fragments of the 3’ terminus of turnip yellow mosaic virus (TYMV; Guerrier-Takada et al., 1988; Green et al., 1988); M, RNA, encoded by bacteriophage 480 (Bothwell et al., 1976a); small hairpin substrates with sequences based on the sequence of various tRNAs (McClain et al., 1987; Forster & Altman, 1990; K. Peck-Miller & S. Altman, unpublished results), and the precursor to 4.5 S RNA (Bothwell et al., 1976b). It is clearly of some interest to elucidate the way in which RNase P can recognize all these different molecules as substrates. Unlike the other substrates that are not precursors to tRNAs, the precursor to 45 S RNA (p4*5S$) from E. coli is the only naturally occurring nontRNA precursor substrate for RNase P that is known to be processed both in vivo and in vitro. In addition, the secondary structure of p45S resembles a long hairpin (Hsu et al., 1984) and is quite different from the secondary structures of the tRNA
portions of precursor tRNAs, which resemble a cloverleaf. Mature 45 S RNA is a stable RNA that interacts with ribosomes during translation (Brown & Fournier, 1984; Bourgaize & Fournier, 1987; Brown, 1987) and which appears to play a role in secretion of certain proteins (Ribes et al., 1990). The study of p45S has now been simplified through the construction of a clone for use in preparation of this RNA in vitro. The kinetic parameters of the reaction with the RNase P holoenzyme (Ml RNA plus C5 protein), or with Ml RNA, its catalytic subunit, alone (Guerrier-Takada et al., 1983), and p45S RNA have been determined, and the effect of deletions in the hairpin structure or 3’terminus of the substrate have provided information on the relative importance of these features for recognition and efficient cleavage by the enzyme. The RNA substrate ~23-4.55, which is identical to wild-type p45S except for the deletion of a single C residue at the second position of the wild-type transcript, was transcribed in vitro from a linearized DNA template using phage T7 RNA polymerase (Fig. 1). Three derivatives of the substrate ~23-455 were also transcribed, each of which contained a stem of only 14 base-pairs in the sequence of the mature molecule instead of 42 base-pairs. These smaller derivatives were designated pS14-CCA, pS14-CCACCC and pS14-CCACCA (Fig. l), and they
7 Present address: Bristol-Meyers Squibb Pharmaceutical Research Institute, 3005 First Avenue, Seattle, WA 98121, U.S.A. $ Abbreviation used: p45S, precursor to 45 S RNA. 1 0022-2836/91/17000145
$03.00/O
0
1991 Academic Press Limited
K. A. Peck-Miller
2
and 8. Altman
~23-4.5s
pS14-CCA
pS14-CCACCC
pS14-CCACCA
5’pppGC_CGCUCUC~u_A~3 U-A G-C U-A g:: GC u uu c
G
G-C G-C C-G U-G C-G U-A G-C
;:g G-U G-C
,P
U G
CAU
Figure 1. Proposed secondary structures of derivatives of ~4.5s RNA. ~23-4.56 is identical to wild-type ~4.58 except for the deletion of a single C residue in the 2nd position of the wild-type transcript (Hsu et al., 1984). The boxed region of ~23-45s indicates nucleotides that were deleted to create pS14-CCACCC. pS14-CCA. pS14-CCACCA and pS14-CCACCC differ in sequence only at the 3’ terminus. The arrow indicates the site of cleavage by MI RNA or RNase P. Each RNA was prepared in vitro from a linearized DNA template using phage T7 RNA polymerase and then gel-purified, as described (Peck, 1990). Sites of cleavage were determined by end-group analysis and by calibration of cleavage products with fragments of known size in sequencing gels (Peck, 1990).
differ from one another with respect to the sequence at the 3’ terminus. The proposed secondary structure of the smaller substrates resembles the small tRNA-like hairpin substrates that are processed efficiently in vitro by RNase P from E. coli (McClain et al.,1987; Forster & Altman, 1990; K. Peck-Miller & S. Altman, unpublished results). Moreover, deletions were positioned such that the sequence GUUCA appeared in the loop region of the small ~23-4.5s derivatives at a position analogous to that of the GUUCG sequence in the small tRNA-like hairpin substrates. These sequences resemble the conserved GTYCR sequence in the T loop of tRNA precursors. A kinetic analysis was made by cleavage of ~23-4.5s and pS14-CCACCC (which have identical 3 termini) by using Ml RNA, the catalytic RNA subunit of RNase P, or RNase P as enzyme (Table 1). Since each kinetic parameter could be reproduced experimentally within a factor of about
2, a difference of threefold or more between kinetic values was considered to be significant. A comparison of the kinetics of cleavage of the precursor to a natural tRNA (pTyr) and a nontRNA (~23-45s) by RNase P from E. coli reveals that the overall efficiency of the reaction (k,,,/K,) with RNase P is similar for each substrate (the relative abundance in the cell of a single tRNA species and 4.5 S RNA is also similar). However, the overall efficiency of the cleavage reaction catalyzed by Ml RNA alone is 11 times greater for pTyr than for ~23-455, and this difference is mostly due to a difference in the K,,, value. For p23-4.5S, the K,,, value of the reaction is decreased Wfold in the presence of C5 protein, the protein subunit of RNase P (Table 1). The effect on the K,,, value is significantly different from that observed with wild-type tRNA precursors, in which case the K, of the reaction catalyzed by RNase P is similar to the K, of the reaction catalyzed by Ml RNA (McClain et al.,
Communications
Table 1 Kinetic parameters of cleavage of substrates derived from pd.53 RNA by Ml RNA and RNase P
( PTY~S p2345s ~814.CCACCC
Ml RNase P Ml RNase P Ml RNase P
XT& 006 903 17.0 62 25 004
&aJKm
lc,,,t
92 66 43 37.0 22.0 290
(x 10-s) 3.3 220.0 93 190.0 8.8 5000
Identical radioactively labeled ([a-32P]GTP) and non-radioactively labeled substrate RNAs were mixed and used for all assays (Peck, 1990). Time points at which the reactions were stopped were adjusted so that measurements were taken on the linear portion of the curve of kinetics of the cleavage reaction. Reactions with Ml RNA as enzyme were carried out in buffer A, and reactions with RNase P as enzyme were carried out in buffer B (described in the legend to Fig. 2). Concentrations of substrates used were optimal for Michaelis-Menten kinetics, and values of K, and V,,. were obtained from Hanes-Woolf plots (Fersht, 1985; Segel, 1975). Each kinetic parameter is the average of results from 2 to 4 independent experiments and each was reproducible within a factor of about 2. t k,,, = mol product/min per mol enzyme. f: From Guerrier-Takada et al. (1988).
1987; Reich et al., 1988; Guerrier-Takada et al., 1988). The distinctive interactions of the hairpin structure of 4.5 S RNA and the cloverleaf structures of tRNAs with the enzyme might explain the difference in the kinetic parameters of their cleavage reactions. Because the K,,, value for the reaction with Ml RNA and wild-type tRNA precursors does not change by more than 50% with the addition of C5 protein, it is likely that the Ml RNA molecule can achieve the correct, or nearly correct conformation for recognition of tRNA substrates without the aid of the protein cofactor. This capability appears to be compromised somewhat in the reaction with p23-45S, as evidenced by the high K,,, value for the reaction with Ml RNA: therefore, C5 protein plays a more significant role in aiding Ml RNA to achieve the necessary conformation of the enzymesubstrate complex with the non-tRNA substrate. Chemical modification of crosslinked complexes of Ml RNA and a tRNA precursor substrate revealed that the substrate may unfold during processing (Knap et al., 1990). Since longer helices are more stable than shorter ones (Freier et al., 1986), the helical structure of ~23-455 may be unfolded less readily than those of tRNA precursors, an effect that could increase the effective K, value of the reaction with ~23-4.5s if unfolding is part of the binding process. The presence of the very basic C5 protein may facilitate unfolding of ~23-4.55, thereby decreasing the effective K,,, value. In fact, in support of these assertions, we found that removal of a large portion of the hairpin of ~23-45s results in a substrate (pSl4-CCACCC) that has a higher k,,, and lower K,,, value for the reaction with Ml RNA compared with the parent substrate (Table 1). The
3
overall efficiency of processing of pSl4-CCACCC by Ml RNA is approximately 30-fold greater than that observed for ~23-4.55. Furthermore, the overall efficiency of cleavage of pSl4-CCACCC by RNase P is slightly greater than that observed for p23-4.5S, mostly as the result of a lower K,,, value for the reaction with pS14-CCACCC. C5 protein has a similar effect on the efficiency of cleavage of each substrate: the decrease in K,,, value of the reaction is much more dramatic than the increase in k,,, value of the reaction in the presence of C5 protein. The portion of the stem that was deleted from ~23-45s is not required for cleavage to take place. Moreover, since pSl4-CCACCC is cleaved at the expected site, it is clear that features of the substrate that are in close proximity to the cleavage site are sufficient for selection of the cleavage site, and that structural features further from the cleavage site are dispensable for this process. The increased overall efficiency of the processing of pSl4CCACCC, compared with that of p23-4.5S, may be due to the fact that pSl4-CCACCC resembles a tRNA precursor in terms of structure more closely than ~23-4.5s does. The helix of 14 base-pairs in the mature portion of pSl4-CCACCC is similar to the helical region of 12 base-pairs near the site of cleavage of tRNA precursors, which results from stacking of the acceptor stem and the T stem (McClain et al., 1987). Since the invariant 3’-terminal CCA sequence of both precursor tRNAs and small tRNA-like precursors is important for cleavage by RNase P from E. coli (Guerrier-Takada et al., 1984, 1989; McClain et al., 1987; K. Peck-Miller & S. Altman, unpublished results), an analysis was made of the importance of the 3’-terminal nucleotides of the substrate pSl4CCACCC. Variants of pSl4-CCACCC were tested that terminated in either a single-stranded 3’ CCA (pSl4-CCACCA) or a double-stranded region containing the “internal” 3’ CCA (pSl4-CCA; Fig. I), instead of the wild-type terminal 3’ CCC sequence. To our surprise, the substrates terminating in CCA were not processed as readily by the enzyme as the substrate that terminated in the wild-type CCC sequence (Fig. 2). Under the conditions used, no cleavage was detected by Ml RNA of pSl4-CCA, although limited cleavage by RNase P was apparent. In the case of pSl4-CCACCA, the extent of cleavage both by Ml RNA and by RNase P was greater than for pSl4-CCA, but the rate of cleavage was still below that of pSl4-CCACCC. Cleavage by Ml RNA is more sensitive to the nature of the 3’-terminal nucleotides of these substrates than is cleavage by RNase P, since the rate of cleavage by RNase P is not as severely affected by changes at the 3’ terminus. A similar pattern was observed with both minimal and full-length tRNA precursor substrates in the presence of C5 protein (Guerrier-Takada et aZ.,1984; Forster & Altman, 1990; K. Peck-Miller & S. Altman, unpublished results). Our results indicate that the 3’.terminal sequence of minimal substrates that resemble ~4.5s RNA is
4
K. A. Peck-Miller
and S. A&man
recursor
3’ Fr,agment -
5’ FIpogment
Figure 2. Cleavage by Ml RNA and RNase P of minimal p45S-like substrates. All substrates were transcribed in vitro in the presence of [c+~‘P]GTP, as described (Peck, 1990). Lanes 1 to 3, pS14-CCA with no enzyme added (lane l), with Ml RNA (lane 2), and with RNase P (lane 3). Lanes 4 to 6 pS14-CCACCC with no enzyme added (lane 4), with Ml RNA (lane 5), and with RNase P (lane 6). Lanes 7 to 9, pS1CCCACCA with no enzyme added (lane 7), with Ml RNA (lane 8), and with RNase P (lane 9). Reactions with Ml RNA were carried out in buffer A (50 mm-Tris. HCl (pH 7.5), 100 mM-NH,Cl, 500 mM-MgCl,) in the presence of 25 ng of Ml RNA for 14 min. Reactions with RNase P were carried out in buffer B (50 mM-Tris . HCl (pH 75), 100 mM-NH&I, 10 mM-MgCl,) in the presence of 3 ng of Ml RNA plus a lo-fold molar excess of C5 for 4 min. All reactions were carried out in a reaction volume of 10 ~1 at 37 “C. The products of the reactions were precipitated with ethanol (in the presence of 20 pg glycogen/ml as carrier) before electrophoresis on a 12% (w/v) acrylamide/7 M-urea gel, then they were visualized by autoradiography. Preparation of Ml RNA has been described (Vioque et al., 1988), and C5 protein was a generous gift from Dr A. Vioque.
important for the cleavage reaction, as it is for the cleavage of tRNA precursors and the minimal substrates derived from them. Despite the fact that a 3’-terminal CCA sequence is present in pS14-CCA, it is a poor substrate (Pig. 2). Therefore, the mere presence of CCA at the 3’ terminus is not enough to promote efficient cleavage of this RNA. The addition of a feature common to tRNA precursors, namely a single-stranded 3’.terminal CCA, to the minimal p4.5S-like substrate pS14-CCA to create pSl4-CCACCA does enhance the rate of cleavage, but not to the same rate as that observed with the analogous substrate that contains the wild-type 3’ terminus (pS14-CCACCC). From these experiments it is clear that some intrinsic feature of the 3’terminal nucleotides of pS14-CCACCC is important for cleavage of this substrate and, presumably, for cleavage of the precursor to 45 S RNA. Additional nucleotides beyond the internal CCA sequence in the double-stranded region are required for efficient cleavage, since cleavage of pS14-CCACCC and pS14CCACCA proceeds with greater efficiency than that of pS14-CCA (Fig. 2). The 3’-terminal CCA of all tRNA precursors is the specific CCA sequence that is important for the cleavage reaction, and the additional internal copies that exist in some substrates are not involved (Sprinzl et al., 1987). Likewise, the internal CCA sequence of p4_5S, which is adjacent to the 3’-terminal CCC, is unlikely to be important for the cleavage by RNase P. Guerrier-Takada et al. (1989) have indicated that the S/-terminal CCA of tRNA substrates interacts with a particular binding site on Ml RNA, although
the absence of the terminal A does not affect the overall rate of reaction (our unpublished results). This proposal may also be relevant to the role of the 3’-terminal CCC of ~4.5s. The interaction with the 3’ end of the substrate, in concert with other features of the substrate, may help to orient the enzyme for correct binding and cleavage of the substrate. This hypothesis is supported by the fact that cleavage of pre-tRNAs (with the exception of pHis) and ~4.5s always occurs in the same position relative to the terminal single-stranded CCA or CCC sequence (Fig. 3).
C C C
A C C
NC -NA
-NB
El .. tf?NA
NC -NA
-NB
El .. 4.5 s
Figure 3. Position of 3’-terminal nucleotides relative to the sites of cleavage of precursor tRNAs and fl5S RNA. Only the nucleotides near the sites of cleavage (arrow) are shown. N, nucleotide; and N, and N, are usually basepaired in tRNA precursors.
Communications We thank the members of our laboratory for helpful discussions. This work was supported by grants from the USPHS (GM19422) and USNSF (DMB8722644) to S.A. References Bothwell, A. L. M., Stark, B. C. & Altman, S. (1976u). PTOC.Nat. Acad. Sci., U.S.A. 73, 1912-1916. Bothwell, A. L. M., Garber, R. L. & Altman, S. (19765). J. Biol. Chem. 251, 7709-7716. Bourgaize, D. B. & Fournier, M. J. (1987). Nature (London), 325, 281-284. Brown, S. (1987). Cell, 49, 825833. Brown, S. & Fournier, M. J. (1984). J. MOE. Biol. 178, 533-550. Fersht, A. (1985). Enzyme Structure and Mechanism, W. H. Freeman and Company, New York. Forster, A. & Altman, S. (1990). Science, 249, 783-786. Freier, S. M., Kierzek, R., Jaeger, J. A., Sugimoto, N., Caruthers, M. H., Neilson, T. & Turner, D. (1986). Proc. Nat. Acad. Sci., U.S.A. 83, 9373-9377. Green, C. J., Vold, B., March, M. D., Joshi, R. L. & Haenni, A.-L. (1988). J. Biol. Chem. 263, 11617-l 1620. Guerrier-Takada, C., Gardiner, K., Marsh, T., Pace, N. & Altman, S. (1983). Cell, 35, 849-857.
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Guerrier-Takada, C., M&lain, W. H. t Altman, S. (1984). Cell, 38, 219-224. Guerrier-Takada, C., van Belkum, A., Pleij, C. W. A. & Altman, S. (1988). Cell, 53, 267-272. Guerrier-Takada, C., Lumelsky, N. & Altman, S. (1989). Science, 246, 1578-1584. Hsu, L. M., Zagorski, J. & Fournier, M. J. (1984). J. Mol. Biol. 178, 509531. Knap, A., Wesolowski, D. & Altman, 8. (1990). Biochimie, 72, 779-790. McClain, W. H., Guerrier-Takada, C. & Altman, S. (1987). Science, 238, 527-530. Peck, K. (1990). Ph.D. thesis, Yale University, New Haven, CT. Reich, C.,‘ Olsen, G. J., Pace, B. & Pace, N. (1988). Science, 239, 178-181. Ribes, V., Romisch, K., Giner, A., Dobberstein, B. & Tollervey, D. (1990). Cell, 63, 591-600. Segel, I. H. (1975). Enzyme Kinetics, John Wiley & Sons, New York. Sprinzl, M., Hartmann, T., Meissner, F., Moll, J. & Vorderwulbecke, T. (1987). NAR 15, r53-rl88. Vioque, A., Arnez, J. & Altman, S. (1988). J. Mol. Biol. 202, 835-848.
Edited by A. Klug