Site selection by Xenopus laevis RNAase P

Cell, Vol. 58, 37-45, July 14, 1989, Copyright 0 1989 by Cell Press

Site Selection by Xenopus laevis RNAase P

Gioia Carrara: Patriria Calandra:t Paoio Fruscoloni:t Margherita Doria: and Glauco l? Tocchini-Valentini’t * Institute of Cell Biology Consiglio Nazionale delle Ricerche Via Romagnosi 18A 00196 Rome Italy t Department of Molecular Genetics and Cell Biology The University of Chicago Chicago, Illinois 60637

Summary Investigation of the mechanism of cleavage site selection by Xenopus RNAase P reveals that the acceptor stem, a 7 bp helix common to all tRNA precursors, is required for cleavage. We propose that Xenopus RNAase P recognizes conserved features of the mature tRNA and that the cleavage site is selected by measuring the length of the acceptor stem. In support of this, we demonstrate that insertion of 2 bp in the acceptor stem of yeast pre-tRNAsbU relocates the cleavage site 2 bases 3’ to the original one. In addition, insertion of 1 bp in the acceptor stem of the endmatured yeast pre-tRNAPhb generates an RNAase P cleavage site: the enzyme produces a mature tRNA with the characteristic 7 bp stem and releases one 5’ flanking nucleotide. Since it has previously been shown that cleavage sites of the splicing endonuclease an? determined by the length of the anticodon stem, RNAase P and the splicing endonuclease apparently use different stems to determine their cutting sites. Introduction Molecules of transfer RNA (tRNA) are synthesized as precursors, which in turn are enzymatically converted to mature tRNA by a series of reactions (Abelson, 1979). The endonuclease RNAase P is responsible for the generation of the 5’ termini of mature tRNA molecules from their precursors (Altman et al., 1988). Avariety of evidence indicates that in both prokaryotes and eukaryotes, a single enzyme can cleave several precursors. Therefore, the enzyme must be dealing directly or indirectly with features that are shared by all the precursors. The precursors probably present a tRNA-like tertiary structure in the mature domain of the molecule, even when an intervening sequence is present. Calculations of free energy minima (Tinoco et al., 1973) and the use of chemical and enzymatic structure-specific probes (Wrede et al., 1979) suggest that all of the S. cerevisiae pre-tRNAs examined have a common tertiary structure (Swerdlow and Guthrie, 1984; Lee and Knapp, 1985). In this structure, the tRNA portion of the precursor maintains the L-shaped conformation,

stabilized by the interaction between the D and T& loops. We obtained a highly purified preparation of RNAase P from Xenopus oocyte nuclei (germinal vesicles or GV). The enzyme requires magnesium ions, and the end groups produced during the cleavage reaction are 5’ P and 3’ OH. How is cleavage site specificity determined? Because the lengths of the leaders and their sequences are not, in general, conserved among different pre-tRNAs (Gold and Altman, 1986; Kassavetis et al., 1989) the main cleavage site determinants must be in the mature domain (Engelke et al., 1985; Leontis et al., 1988). To obtain information on the features of the pre-tRNAs recognized by E. coli RNAase P McClain et al. (1987) produced mutant precursors that lack specific domains of the normal tRNA sequence. The smallest tRNA precursor that was cleaved efficiently retained only the helical segment of the amino acid acceptor stem and the T stem-loop. The implication of these results is that the cut-down substrate contains determinants for precursor recognition and cleavage. Utilizing yeast pre-tRNAy and a series of its variants constructed by in vitro mutagenesis, we show that the acceptor stem, a 7 bp helix common to all precursors, is required for cleavage by the eukaryotic RNAase P derived from Xenopus oocytes. There is evidence indicating that, once positioned on the precursor, another enzyme utilizing pm-tRNA as substrate (the splicing endonuclease) determines the cleavage sites by measuring length along the anticodon stem (Reyes and Abelson, 1988; Mattoccia et al., 1988). We propose that Xenopus RNAase P interacts with the precursors via some conserved features of the mature domain and that the cleavage site is selected by measuring length along the acceptor stem. In support of our hypothesis, we demonstrate that the insertion of 2 bp in the acceptor stem of yeast pretRNAp alters the cleavage site in a predictable way. The site is relocated 2 bases 3’to the original one. In addition, we show that the insertion of 1 bp in the acceptor stem of the end-matured yeast pre-tRNAPhe generates a cleavage site for Xenopus RNAase P The enzyme produces a mature tRNA with the characteristic 7 bp stem and releases one 5’ flanking nucleotide. Results The Acceptor Stem Is Required We have observed that the Xenopus RNAase P can recognize and specifically cleave a variety of different precursors, such as C. elegans pre-tRNALeU (Tranquilla et al., 1982), Xenopus pre-tRNAMet (Koski and Clarkson, 1982) and yeast pre-tRNATv, pre-tRNAp (Beckman et al., 1977) and pre-tRNAPhe (Reyes and Abelson, 1987). In the studies reported in this paper, we chose to concentrate on yeast pre-tRNAp and pre-tRNAPhe, class 2 and class 1 precursors; respectively.

S.cererisiae

UC-H

pm-tRNAp

i U A A

(6)

(A) tpG.C G.C U.A ” . Am SG . C lJ.G

‘;Lp A G U

C

Anticodon

-A

A AU

Figure 1. Secondary Structure Model of Wild-Type Pre-tRNAp with the Two Leaders, A and 0, at its 5’ End and the U Cluster at its 3’ End Nucleotides are numbered according to the yeast tRNA system. The darkened triangle indicates the single cut generated by RNAase P endonuclease. Splice junctions are indicated by arrows.

Transcription of the yeast tRNAp gene in GV extracts (Mattoccia et al., 1979) starts at two sites: A-4 and A-3 (Figure 1). W h e n a mixture of the two 5’ 32P end-labeled was incubated with Xenopus RNAase P pre-tRNAsp (Fraction IV; see Experimental Procedures), two oligonucleotides (A and B) were produced, 4 and 3 U long, re-

Figure 2. Identification of the 5’ Terminal Oligonucleotides Produced by RNAase P Cleavage of Wild-Type Pre-tRNAp

12345676 .,.-t..A~”

A.

spectively (Figure 2A). The A and B leaders have mobilities consistent with the expected sequences, which lack phosphate at the 3’ termini. Analysis of the two leaders was carried out on a polyacrylamide sequencing gel (Figure 28). Oligonucleotides A and B comigrate, respectively, with the tetra and trinucleotides that result when the 5’ 32P end-labeled precursors are digested with ribonuclease Pl (an endonuclease that forms mononucleoside-5’-phosphates), thereby confirming the presence of 3’ OH termini (Figure 28; lanes 1 and 12). The sequence of the A leader is AAGUon and the sequence of the B leader is AGUoH. Figure 28 shows the ladder sequence analysis: the oligonucleotides were digested with alkali (lanes 2 and 8) Up, an adenosinespecific nuclease (lanes 3 and lo), and T1, a guanosine-specific nuclease (lanes 4 and 9). Two-dimensional RNAase T, fingerprint analysis of the unique large product of RNAase P cleavage indicates that its 5’end is identical to the 5’end of mature tRNA (Otsuka et al., 1981) (data not shown). An SP8 transcript of the yeast tRNAy gene, characterized by a leader 37 nucleotides long, is cleaved at the usual site by the Xenopus RNAase R albeit at a reduced rate (G. Carrara and D. Civitareale, unpublished data). These results demonstrate that, as expected, the RNAase P cleavage site is independent of leader length. Given that leader length does not affect the location of the cleavage site and that in general there are no sequences conserved in the flanking regions of the tRNA genes (Gold and Altman, 1986; Kassavetis et al., 1989) the determinants of the RNAase P cleavage site must be located in the mature domain. To investigate the possible role of the acceptor stem, a

0

(A) 3 x 10e3 pmol of 5’ 32P end-labeled prewas incubated at 3pC under stantRNAy dard conditions both with (E) and without (C) RNAase P enzyme, for 30 min (lanes 3 and 5) and 1 hr (lanes 4 and 6). The same precursor was partially digested with alkali (lanes 1 and 7) and RNAase T, (lanes 2 and 6). Samples, stopped as described in Experimental Procedures, were run on a 16% polyacrylamide-7 M urea sequencing gel. The A and B leaders are indicated. (6) Gel-purified A and 6 leaders (A) were subjected to limited hydrolysis with RNAase U2 (lanes 3 and IO), RNAase T, (lanes 4 and 9) and alkali (lanes 2 and 6). C represents untreated 5’ sap end-labeled leader RNAs (lanes 1 and 12). 5’ssP end-labeled pre-tRNAp was subjected to partial digestion with alkali (lanes 7 and 11) RNAase Tr (lane 5), and nuctease Pl (lane 6). After separation of the products by 20% polyacrylamide-7 M urea sequencing gel electrophoresis, the sequence was read directly from the autoradiograph.

Eukaryotic 39

RNAase P Measuring

Mechanism

C.

A.

: PP....

;

.

.

h-P.*

l :

l. . :=:-: .-. .-. .-.

0.

‘..

.-. .-. .-. . 0-0 . . . . . . . .

... :

:;A::

‘::~*

l ...*

Figure 3. Cloverleaf Representation tRNAp Precursor Variants

.0* .: . .: : 0-U ;z;

.0*

...

0..

-0.

.

._

.:.J.f:*

l

.-. ..*l . . .-. .-.

*

.-..*. . .-. .c>. l -• ‘.I:.* .-. .-. .-.

..’

l

of Yeast

The nucleotides indicated in A-D show the base substitutions present in the variants. The nucleotides indicated in E show the base insertions in the mutant pre-tRNAp @Vacc.s. The circles in F indicate the deleted nucleotides in the mutant pre-tRNAy EAA. The symbol V refers to an insertion mutation and the symbol A refers to a deletion. The new RNAase P cleavage site in the mutant pretRNALeU E Vaccs IS mdlcated by a darkened:ria!gl!

l .

.

0.

.

.

. .

.

F. .0* . . : : :

PeP-. -.

.

. . ..* *.*.*

: t ., .t .*--

: .-. .-. .-. .-. .-. .-.

0-a

::::: =:--**

. .

-. . *

. &f~ .-. .-. .** .-..*. . .-. .+. .-. .*‘,.. .-. l .,,*. .-. .-. l . .. . . . . . E.4 a

7 bp helical structure common to all precursors, we produced a series of substitution mutants of pre-tRNA$eU. Time courses of cleavage of wild-type and mutant precursors (Figures 3A, 38, 3C, and 3D) were compared to see how changes in the mutants affect cleavage (Figure 4). Comparisons of the initial rates of precursor utilization showed that the elimination of 1 bp (GlA, GlU) causes a 50% reduction in cleavage; more extensive disruption of helicity (acc.s.Sl and accsS2) renders the mutant precursors practically unsusceptible to the enzyme. In the double mutant (acc.s.Sl-2) the ability to form a stem is retained and the initial rate of utilization is identical to that of the wild type. The location of the cleavage site was verified by sequencing the leader and the mature product (data not shown). An intact acceptor stem appears to be required for RNAase P cleavage. Determination of Cleavage Site Specificity Having shown that an acceptor stem is required for RNAase P cleavage, we continued to investigate its role in the reaction, testing whether RNAase P specificity

might be determined by the distance from the contact points in the mature domain to the cleavage site. This distance is fixed because all tRNAs have the same number of base pairs in the acceptor stem. It would follow that an increased number of base pairs in the acceptor stem should change the distance and alter the cleavage site in a predictable way. Therefore, we produced the mutant pre-tRNAy ($)Vacc.s. (Figure 3E) by inserting 2 bp in the acceptor stem. Incubation of a tRNA gene in GV extract results in a simultaneous transcription and processing reaction. Although the pre-tRNA p ( ti ) Vacc.s. is 4 bases longer than wild-type pre-tRNA$eU, the mature tRNA that forms, is of the correct size (though produced in reduced amounts) (Figure 5A). The sequence of the mature mutant tRNA (data not shown) indicates that the intron has been excised precisely as expected since the splice sites are determined by the length of the anticodon stem (Reyes and Abelson, 1988; Mattoccia et al., 1988). The sequence also indicates that the acceptor stem is 7 bp long. These results suggest that a mechanism to keep the length of the acceptor stem

Cell 40

C

MT

1’

2’

3’

5’

8’ 15’

A.

suggest that some factor(s) is lost during RNAase P purification. Deletion of the Extra Arm Does Not Affect 5’ Processing The reference points in the mature domain remain undetermined. It is clear, however, that RNAase P must be dealing with features shared by all the precursors. Because most tRNAs do not have an extra arm (a structural element common to class 2 pre-tRNAs such as pretRNAp), it is unlikely to be important in 5’ processing. We have observed that its deletion does not affect RNAase P Figure 4 shows that the mutant pre-tRNAp EA A (Figure 3F) obtained by deletion of nucleotides 79-86 is perfectly processed by RNAase I?

123

5

8 Minute

Figure 4. Kinetics of RNAase P Cleavage Precursors

15

of Wild-Type and Mutant

(A) Time course for RNAase P cleavage of [a-32P]GTP-labeled wildtype pretRNAp (1.5 x 10m3 pmol). Time 0 (C) is the untreated precursor. MT markers refer to products of simultaneous transcription and processing reaction of wild-type tRNAp (Otsuka et al., 1991). (B) Comparison of the initial rates of RNAase P cleavage of different precursors. Identical time course experiments as in (A) were run for individual mutant precursors. Percentages of uncleaved precursors are plotted at each time point. There is a significant difference between the and that of the wild-type rate (2.7 x 1O-4 pmol/min-t) ( H) pm-tRNAp GlA (M) and pm-tRNAp GIU (M) mutants (1.35 x 10W4pmol/min-l). RNAase P cleavage was practically nonexistent in the case of pre-tRNAp acc.s.Sl (M) and preand acc.s.S2 (-). Pre-tRNAp EA A ( l -e) tRNAp ) precursors show the same kipm-tRNAp acc.s.Sl-2 (M netic profile as wild-type pm-tRNAp.

constant at 7 bp in mature tRNA operates both in 5’ and 3’ processing (Carrara et al., unpublished data). Therefore, in the case of RNAase P, the insertion of 2 bp in the acceptor stem should cause a 2 base shift of the cleavage site. The shift was in fact observed when pretRNAp (z)Vacc.s., synthesized in vitro, was incubated in GV extracts. At the 5’end of wild-type mature tRNA, the sequence is pGGUUGUU, and at the Blend of the mutant it is pUU(CU)GUU (the inserted bases are indicated in parentheses) (Figure 58). These findings show that, as a result of the insertion of 2 bases, the cleavage site is relocated 2 bases 3’ to the original one. Purified RNAase P cleaves the pre-tRNe (E)Vacc.s. at the shifted site only 66% of the time. Forty percent of the time the enzyme cleaves the precursor 1 base 3’ to that site. One possible way to interpret these results is to

Generation of an RNAase P Cleavage Site in End-Matured Pre-tRNAPhe If the RNAase P cleavage site is determined by the length of the acceptor stem, it should be possible to generate a cleavage site in an end-matured pre-tRNA by inserting a base pair in the acceptor stem. To verify this possibility, we used the yeast tRNAPhe gene produced by Reyes and Abelson (1987) (Figure 6). The T7 transcript of this synthetic gene consists in an endmatured pre-tRNA containing a 19-base-long intervening sequence. We synthesized the pre-tRNAPM in vitro in the presence of cold ribotriphosphates. After alkaline phosphatase treatment, the primary transcript was labeled at its 5’ end with T4 polynucleotide kinase. As expected, RNAase P did not cleave the end-matured molecule (Figure 7, lanes 2 and 3). Incubation with RNAase P of 5’ 32P end-labeled mutant pre-tRNAPhe, which is characterized by the insertion of a single base pair in the acceptor stem (AUVacc.s., Figure 8) caused the release of one nucleotide at the 5’ end of the molecule (Figure 7, lanes 8 and 9). The nucleotide comigrates both with the product of a complete digestion of an [a-32P]GTP-labeled RNA molecule with snake venom phosphodiesterase, a 3’exonuclease that forms mononucleoside-5’-phosphates (Figure 7, lane 6) and with the product of the extensive digestion of 5’ 3*P end-labeled pre-tRNAPhe and 5’ 3*P end-labeled pre-tRNAPhe AUVacc.s. (Figure 7, lanes 1 and 7) with ribonuclease Pl, an endonuclease which forms mononucleosided’-phosphates. We conclude that the nucleotide can only be pGoH. After alkaline phosphatase treatment, the large product of the cleavage reaction was labeled at the 5’ end with T4 polynucleotide kinase. Comparison of the partial digestion of 5’ 3*P end-labeled pre-tRNAPhe AUVacc.s. (Figure 7, lanes 4 and 5) and of 5’ 32P endlabeled RNAase P large product (Figure 7, lanes 12 and 11) with alkali and T, confirmed that pGou had been removed. These results indicate that the insertion of a base pair in the acceptor stem of an end-matured pre-tRNA converts the molecule into a substrate for RNAase P These findings lend support to our hypothesis that the cleavage site is determined by the length of the acceptor stem. It should be noted that the precursor used in these experiments is efficiently cleaved despite the fact that it is unmodified (Leontis et al., 1988). The synthetic precursor

Eukaryotic 41

RNAase P Measuring

Mechanism

B. 1

234S6

78

1

9

unspliced precursors

-U

;I

-U

UCL

,tJ -c

-tRNA U-

44

U-

Figure 5. Transcription and Processing

Products of Wild-Type and Mutant [($)Vacc.s.]

tRNAp

Genes

(A) In vitro transcription and processing of wild-type and mutant [(t:)Vacc.s.] #INAp genes. DNAs were transcribed in GV extracts of Xenopus oocytes and the products separated on a 10% polyacrylamide gel (Saldi et al., 1983): Wr, lane 2; ($)Vacc.s., lane 1. Individual bands of wild-type tRNAp gene simultaneous transcription and processing reaction (lane 2) were previously characterized by fingerprinting analysis (Otsuka et al., 1981). (8) Sequence analysis of 5’ termini of wild-type and (t:)Vacc.s. tRNAs. Wild-type and mutant tRNApacc.s. pre-tRNAs were synthesized using our in vitro system (see Experimental Procedures) in the presence of the four nonradioactive ribonucleotide triphosphates (0.2 m M of ATP, CTR and UTP and 15 pM of GTP). The gel-purified pre-tRNAs were then incubated in GV extract in the presence of cold ribotriphosphates. After 1 hr at 22oc, the reaction was stopped and the products were analyzed by gel electrophoresis as described, and the bands corresponding to tRNAs were purified (Baldi et al., 1983). After treatment with alkaline phosphatase, they were labeled with [Y-~P]ATP and partially digested with alkali (lanes 1 and 8) RNAase T, (lanes 3 and 7) RNAase Us (lanes 4 and 5) and RNAase 8. cereus (lane 8). C represents untreated end-labeled tRNAs (lanes 2 and 9). Each reaction contained 3 x 10e3 pmol of end-labeled tRNA; samples were electrophoresed on a 20% polyacrylamide-7 M urea sequencing gel. The nucleotides indicated in the margin stand for the 5’ terminus tRNA sequences; the two bases indicated in parentheses are the inserted nucleotides in the tRNAp @)Vacc.s. mutant.

also differs from the natural precursor because it does not contain a 5’ triphosphate terminus (pppG). Apparently, this difference is not important for RNAase P cleavage. The Splicing Endonuclease and RNAase P Measure Different Stems The insertion of 1 (GCVant.s.) and 2 ((X$)Vant.s.) bp in the anticodon stem of the tRNAPhe gene (Reyes and Abelson, 1986 and Figure 6) does not generate an

RNAase P cleavage site because pGo~ is not released (Figure 8, lanes 6 and 14). It has already been reported that the cleavage sites of the splicing endonuclease are determined by the length of the anticodon stem (Reyes and Abelson, 1988; Mattoccia et al., 1988). It thus appears that the two endonucleases utilize different stems to determine their cutting sites (Figure 9). At present, it is not clear whether E. Coli RNAase P operates in the same way as the Xenopus en-

S. cerevisiae

pre-tRNA

AOH C C

Phe

12345678910 WT

IP(

c” A

U.A GACAC ciiciri; C U c

U A

UcA CUCG

U G

cici:

AU Vaccs. E E 1 OH-T, lsvl F-4 E E I Ptl

c uu

c

,. ’ ”

GCA ’

AC C-G

\C.& (Reyes

L Abelson)

Figure 6. Secondary

A

*

U-G c-c A4 A.U 1 c.c A A A A AA

Structure of Yeast Pre-tRNAPhe

The figure shows variants of the mature domain, constructed so as to alter the length of either the acceptor stem or the anticodon stem. -I%,

-PA,,

zyme (Burkard et al., 1988). A measuring mechanism for the E. Coli enzyme was hypothesized by Bothwell et al. (1976).

I)

-pCGGp

e

-PC=P

Discussion -P=P

In both prokaryotes and eukaryotes, a single RNAase P can recognize and cleave several precursors (Altman et al., 1986). There is good evidence that the tertiary structure of tRNA precursors, even when an intron is present, is dominated by the mature domain. Structure probing of a number of tRNA precursors (Wrede et al., 1979) has shown that both secondary and tertiary interactions typical of mature tRNA take place in the precursor (Swerdlow and Guthrie, 1984; Lee and Knapp, 1985). In general, the interactions of RNA molecules with enzymes bear greater resemblance to protein-protein binding than to protein-DNA binding. The analysis of simplified systems has shown that both the conformation and a few specific, highly conserved nucleotides are recognized (Wickens and Dahlberg, 1987). Presumably, like the splicing endonuclease (Reyes and Abelson, 1988; Mattoccia et al., 1988), eukaryotic RNAase P binds to the mature domain by way of multiple interactions. It seems likely that the recognition elements are to be found among the conserved bases (Engelke et al., 1985; Leontis et al., 1988). As most tRNAs do not have an extra arm, it is unlikely to be a contact point. W e have in fact observed that deletion of the extra arm in the pre-tRNAp does not affect the specificity of RNAase P cleavage. W e envisage that RNAase P, like the splicing endonuclease (Baldi et al., 1986) acts in two steps: specific recognition and binding

-PCP

Figure 7. RNAase P Cleavage of Pre-tRNAPhe and Pre-tRNAP& AUVaccs. RNAase P Activity on Pm-tRNAPhe and Pre-tRNAPhe AU-

Vaccs. (A) Products of RNAase P activity. 3 x 1O-3 pmol of 5’=P end-labeled wild-type pm-tRNAPhe (lanes l-3) and mutant pm-tRNAP” (lanes 4 and 5; 7-9) were incubated with RNAase P enzyme (E): Fraction IV (lanes 2 and 8) and Fraction V (lanes 3 and 9). The precursors were also extensively digested with endonuclease Pl (lanes 1 and 7). Sendlabeled pre-tRNAPhe AUVaccs. was subjected to partial digestion with alkali (lane 4) and RNAase T, (lane 5). pGon marker was obtained by a complete digestion of [a-32P]GTP uniformly labeled RNA with snake venom phosphodiesterase (lane 6). pAon marker was obtained by a complete digestion of 32P end-labeled pre-tRNA!$” with endonuclease PI (lane 10). The 5’ pre-tRNAP” AUVacc.s. RNAase P product (lanes 8 and 9) migrated nearly 2 bp more slowly than comparable length alkali and RNAase T, products (lanes 4 and 5) as a result of the lack of a 3’ terminal phosphate group. (B) Sequencing analysis of the large product (‘) of the cleavage reaction of pre-tRNA phe AUVacc.s. 5’ ssP end-labeled pre-tRNAPhe AUVacc.s.was incubated with RNAase P under standard conditions. The large product of the reaction was eluted from the gel and, after alkaline phosphatase treatment, end-labeled with [Y-~P]ATP The labeled molecules were subjected to partial digestion with alkali (lane 12) and RNAase T, (lane 11). Following separation of the products by 16% polyacrylamide-7 M urea gel electrophoresis, the sequence was read directly from the autoradiograph.

Eukaryotic 43

RNAase P Measuring

Mechanism

123456789 A u V acc.s. ’ c

PGP-

WT

GCVant.s.

E P, ‘E T, OH- c

-

Figure 8. Incubation of RNAase P with 5’ 32P End-Labeled Pre-tRNAPhe Variants

10 11 12 13 1415 16 17

’

c

on-q

(s)%t.*. E ‘E

T,

OH-C

’

The transcripts were pre-tRNAPhe wt (lanes 10-13) pre-tRNAPhe AU9acc.s. (lanes I-5) pm-tRNAP” GCVant.s. (lanes 6-9) and pretRNAPhe (!=$)Vant.s. (lanes 14-17). C represents untreated end-labeled precursors (lanes 1,9, 10, and 17). E represents RNAase P digestion (lanes 4, 6, 13, and 14). Precursors were partially digested with alkali (lanes 2.8, Il. and 16) RNAase T, (lanes 3, 7, 12, and 15) and nuclease PI (lane 4).

I II

of the precursors, and cleavage to remove the leader. How is cleavage site specificity determined? Given that the sequences of the leaders and of the acceptor stems of different pre-tRNAs are minimally conserved, it is clear that nucleotide sequence per se does not play a major role in cleavage site recognition. The length of the leader varies among different pre-tRNAs (Gold and Altman, 1988; Green and Vold, 1988; Kassavetis et al., 1989) and we show here that the variants of a single precursor, characterized by different leader lengths, are all cleaved at the same site by Xenopus RNAase l? Both the length and the location of the acceptor stem are conserved in all precursors. Once the enzyme is bound through interactions with conserved elements of the mature domain, this geometry ensures that the alignment of the cleavage site with RNAase P wilJ be’the same. We propose that after RNAase P has recognized features common to all precursors and positioned itself on the molecule, it cleaves at a fixed distance from the reference points in the mature domain.

We report in this paper that a 7 bp acceptor stem is required for pre-tRNA 5’end-maturation; disruption of helicity renders the mutant precursors particularly unsusceptible to the enzyme. A mutant pre-tRNAp characterized by the insertion of 2 bp in the acceptor stem, although 4 bases longer than the wild-type precursors, is processed in vitro to a mature tRNA of the correct size. The RNAase P cleavage site is relocated 2 bases 3’to the original one. Finally, we demonstrate that the insertion of 1 bp in the acceptor stem of end-matured pre-tRNAPhe converts the molecule into a substrate for RNAase P generating a cleavage site. The enzyme produces a mature tRNA with the characteristic 7 bp stem and releases one 5’ flanking nucleotide. It has already been reported that the cleavage sites of the splicing endonuclease are determined by the length of the anticodon stem (Reyes and Abelson, 1988; Mattoccia et al., 1988). It thus appears that the two endonucleases utilize different stems to determine their cutting sites.

Cell 44

AT-3’, and the same procedure described for the construction of the double mutant acc.s.Sl-2. The first oligonucleotide, complementary to the coding strand from position -6 to position 12, was used to insert 2 bases between positions 4 and 5 of the wild-type acceptor stem. The second oligonucleotide, complementary to the coding strand from positions 102 to position 119, was used to insert 2 bases between positions 109 and 110 of the wild-type acceptor stem. The IEmer oligonucleotide 3’-TGAGTCCATACGTTCTCA-5’, complementary to the noncoding strand from position 70 to position 95 but carrying the deletion of the nucleotides AGCATTCTfrom position 79 to position 66, was used to delete the extra arm of the tRNAp gene. The EA A mutant was derived from the pTGEM2 plasmid (Mattoccia et al., 1966) (Figure 3F). Synthesls of tRNA Precursors Germinal vesicle (GV) extract from stage-six oocytes was prepared according to Mattoccia et al. (1979). Transcription of wild-type and mutant tRNAp genes and processing of their precursors in GV extracts were carried out as previously described (Baldi et al., 1963; Gandini-Attardi et al., 1965). To prepare 5’end-labeled pre-tRNAs, purified unlabeled precursors were dephosphorylated and labeled with “P using T4 polynucleotide kinase and [T-32P]ATP (3009 Cilmmol, Amersham). After labeling, the precursors were ethanol-precipitated and repurified on 10% polyacrylamide-l M urea gels. The wild-type and mutant tRNAPhe precursors were synthesized utilizing T7 RNA polymerase according to Reyes and Abelson(1966).

Figure 9. Model for Splice Site Recognition by Splicing Endonuclease (Reyes and Abelson, 1966) and by RNAase P The precursor tRNA is shown with its leader, trailer, and intervening sequence. Both splicing endonuclease and RNAase P are proposed to interact with some conserved parts of the mature domain. Once anchored, they measure fixed distances relative to the mature domain and cut, each according to its own specificity.

Experimental

Procedures

Plasmids Plasmid pTC was constructed by subcloning the 2.5 kb EcoRI-DNA fragment containing the tRNAp gene from pJB2K (Beckman et al., 1977) into pBR322 at the EcoRl site as described by Johnson et al. (1960). Plasmid pTGEM2 was constructed by subcloning the 165 bp Rsal-DNA fragment containing the tRNAp gene from pTC plasmid into pGEM1 (Promega) at the Smal site. PUC13p” and the M13Phe AUVacc.s., GCVant.s., and (!$)Vant.s. variants were gifts from J. Abelson (Reyes and Abelson, 1966). Mutagenesis The oligodeoxynucleotides were synthesized utilizing the Beckman system 1 plus automatic synthesizer. The mutagenesis procedure has been previously described (Baldi et al., 1963). To mutagenize the nucleotide Gi, we synthesized one 17-mer oligonucleotide complementary to the noncoding strand from position -6 to position 9 of the tRNAp gene (plasmid pTGEM2). The oligonucleotide 3TTTATTCA:CAACAAAC-5’ was a mixed probe that allowed us to substitute the nucleotide Gl with A(GlA) or U (GIU) (Figure 3A). The 19-mer oligonucleotide Y-CAAATAAGATCGTGTTTGG-3’ complementary to the coding strand from position -9 to position 10 was used to construct the mutant acc.s.Sl (Figure 38). The 14mer oligonucleotide 5’CTTAGCACGATATAATTTT-3’ complementary to the coding strand from position 104 to position 122 was used to construct the mutant acc.s.SZ (Figure 3C). To obtain the double mutant acc.s.Sl-2, we cut both the single mutants with Bgll and Hpal restriction enzymes. The purified Bgll-Hpal fragment from acc.s.Sl containing the 5’ half of the tRNA!$” gene and the purified Hpal-Bgll fragment from acc.s.S2 containing the 3’ half of the tRNAp g ene were ligated with the T4 DNA ligase. Plasmid DNA from recombinant colonies in which ampicillin resistance had been restored were checked for the presence of the double mutant acc.s.Sl-2 gene (Figure 30). To obtain the double mutant (tz)Vacc.s. (Figure 3E), we used the two 29mer oligonucleotides 5’-ATAAGTGGTTCTGTTTGGCC3’ and 5’CTCTTAGCAGAACCAATA-

Purification Pmcedure All procedures were carried out at 4OC. The nuclear extract (27 ml, 106 mg) was prepared from 170 ml of collagenase-treated oocytes and frozen at -70% until use. The extract (Fraction I) was dialyzed against TEMG buffer (50 m M Tris-HCI [pH 7.51) 1 m M EDTA, 1.4 m M P-mercaptoethanol and 16% (v/v) glycerol) and applied to a 40 ml phosphocellulose column equilibrated with TEMG buffer (pH 7.5). The column was washed with the equilibration buffer and eluted with a 600 ml nonlinear gradient from 0 to 1.5 M KCI in the same buffer (flow-rate 50 ml/hr). Both the J’and 5’ processing endonucleases eluted together between 0.064 and 0.3 M KCI. Peak fractions of activity were pooled and dyalized against 50~01 of JB (7 m M MgCIz, 0.1 m M EDTA, 2.5 m M dithiothreitol, 10% (v/v) glycerol, and 10 m M HEPES [pH 7.51) containing 35 m M NH&I. The dialyzed sample (Fraction II) was applied to a column (1 x 57 cm) of DEAE-Sephadex A 25 equilibrated in JB 35. The column was washed with 3 column vol of equilibration buffer. The enzymes eluted as separated activities by a 250 ml linear gradient from 35 m M to 1.2 M NH&I in the same buffer. RNAase P eluted at 0.25 M NH&I (Fraction Ill). The active fractions were dialyzed against JB 35 and applied to a 5 ml column of DEAE-Sephacel equilibrated with the same buffer. The column was washed with the equilibration buffer and eluted with a 25 ml linear gradient from 35 m M to 1.2 M NH&I in the same buffer. RNAase P activity (Fraction IV) eluted at 0.3 M NH&l and the enzyme containing fractions were frozen at -70°C. Aliquots of Fraction IV were applied onto 5 ml glycerol gradients (15%-30% glycerol in JB 150 m M NH&I) and centrifuged for 15 hr at 46 K in SW 55.1 rotor at 4OC. A total of 24 fractions was collected from the bottom of the tube. The RNAase P active fractions (Fraction V) were added to BSA (Boehringer Mannheim) at 200 uglml and stored at -7OOC. Assay for RNAase P Activity In 20 VI of JB 70 m M NH&I, the standard reaction mixture contained 1.5 x 10m3 pmol of gel-purified pre-tRNA substrate and 10 nl of enzyme; reactions were incubated for 30 min at 3pC and stopped by addition of SDS to 0.5% and proteinase K to 0.1 mglml. After incubation for 30 min at room temperature and the addition of 1 pg of yeast tRNA and NaCl to 0.2 M, the samples were precipitated with 3 vol ethanol at -7ooC for 30 min. The level of RNAase P activity used in time course experiments utilizing pre-tRNA mutant precursors (Figure 4) was previously determined using a wild-type pre-tRNA precursor to obtain the linear rates of appearance of the cleavage products in the first minutes of the reaction. At each time point, aliquots were withdrawn and the RNA species were analyzed as previously described (Baldi et al., 1963). Quantitation of the products of the reactions was accomplished by excising gel slices containing radioactively labeled RNA and determining Cerenkov counts (30% counting efficiency).

Eukaryotic 45

RNAase P Measuring

Mechanism

RNA Sequence Analysis The 5’ end-labeled RNA species (Baldi et al., 1983) were partially digested with alkali, with endonucleases Pi (Sigma) and B. cereus (BRL). and under fully denaturing conditions, with RNase Tr (BRL) and RNAase U2 (BRL) according to Donis-Keller et al. (1977). The reactions were stopped by addition of urea to 7 M, glycerol to 10% (v/v), and xylene cyanol and bromophenol blue to 0.05 (w/v) and immediately loaded on and electrophorized through a 18% polyacrylamide-7 M urea sequencing gel (Maxam and Gilbert, 1977); 20% polyacrylamide-7 M urea gels were used to improve the resolution of the very short fragments.

Acknowledgments We are grateful to J. Abelson and E. R Geiduschek for critical reading of the manuscript and to V. M. Reyes and J. Abelson for the gift of the synthetic tRNAP” and the variants tRNAP” genes. We wish to thank G. Guidi and G. Di Franc0 for excellent technical assistance, and A. Sebastian0 for typing the manuscript. This work was supported in part by “Progetto Finalizzato Oncologia: by “Progetto Finalizzato lngeg neria Genetica e Basi Molecolari delle Malattie Ereditarie,” by Progetto Finalizzato Biotecnologie e Biostrumentazione, and by an EniChem grant. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked bdverfisemenf” in accordance with 18 USC. Section 1734 solely to indicate this fact.

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Received March 7, 1989; revised April 20, 1989

Maxam, A. M., and Gilbert, W. (1977). A new method for sequencing DNA. Proc. Natl. Acad. Sci. USA 74, 580-584.

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