Several regions of a tRNA precursor determine the Escherichia coli RNase P cleavage site

J. Mol. Biol. (1992) 227, 1019-1031

Several Regions of a tRNA Precursor Determine Escherichia colt’ RNase P Cleavage Site

the

Staffan G. Svhrd and Leif A. KirsebomjDepartment of Microbiology Box 581, Biomedical Center, S-751 23 Uppsala, (Received 6 April

1992; accepted 25 June

Sweden

1992)

The RNase P cleavage reaction was studied as a function of the number of base-pairs in the acceptor-stem and/or T-stem of a natural tRNA precursor, the tRNATY’Su3 precursor. Our data suggest that the location of the Escherichia coli RNase P cleavage site does not depend merely on the lengths of the acceptor-stem and T-stem as previously suggested. Surprisingly, we find that precursors with only four base-pairs in the acceptor-stem are cleaved by Ml RNA and by holoenzyme. Furthermore, we show that both disruption of base-pairing, and alteration of the nucleotide sequence (without disruption of base-pairing) proximal to the cleavage site result in aberrant cleavage. Thus, the identity of the nucleotides near the cleavage site is important for recognition of the cleavage site rather than base-pairing. The important nucleotides are those at positions - 2, ~ 1, + 1, + 72, + 73 and + 74. We propose that the nucleotide at position + 1 functions as a guiding nucleotide. These results raise the possibility that Mg2+ binding n ear the cleavage site is dependent on the identity of the nucleotides at these positions. In addition, we show that disruption of base-pairing in the acceptor-stem affects both Michaelis-Menten constants, K,,, and I%,,,.

Keywords: catalytic

RNAs;

RNase P; substrate tRNA mutants

1. Introduction The maturation of the 5’.termini of the tRNAs is the result of cleavage by RNase P. The catalytically active subunit of Escherichia coli RNase P is an RNA (Ml RNA). The other subunit of RNase P in E. coli, C5 protein, is not necessary for cleavage in vitro but is absolutely required for cleavage in vivo (Altman et al., 1986 and references therein). To understand the function of RNase P it is of importance to elucidate how this enzyme recognizes its substrates. It is known that the tertiary folding of the tRNA moiety of a tRNA precursor molecule plays a significant role in the enzyme-substrate interaction (Altman et al., 1974; Kirsebom & Altman, 1989). Earlier studies have shown that the amino acid acceptor-stem in a tRNA precursor is an important determinant in this process (McClain et al., 1987; Burkard et al., 1988; Green & Vold, 1988; Nichols et al., 1988; Guerrier-Takada et al., 1989; Helm & Krupp, 1992; Kirsebom & SvSird, 1992). Also, previous reports have demonstrated that disruption of base-pairing at or near the site of cleavage can affect the kinetics of cleavage and 1‘ Author to whom all correspondence should be addressed. 0022-2836/92/2OlOl9-13

$08.00/O

recognition;

tRNA

processing;

location of the cleavage site (Burkard et al., 1988; Green & Vold, 1988; Nichols et al., 1988; Kirsebom & SvLd; 1992). Furthermore, it has been suggested that the location of the site of cleavage for RNase P is determined by the length of the T-stem and acceptor-stem of a tRNA precursor (Carrara et al., 1989; Kahle et al., 1990). In an attempt to understand the significance of the primary structure and length of the acceptor-stem of a tRNA precursor in the cleavage reaction we decided to study cleavage by E. coli RNase P as a function of the number of base-pairs in the acceptor-stem of the tRNATYTSu3 precursor. Our results show that the locaton of the E. coli RNase P cleavage site does not depend in a simple way on the length of the acceptor-stem and/ or T-stem. Rather the results suggest that several regions of a tRNA precursor are important in the selection of the cleavage site. We also show that precursors with only four base-pairs in their acceptor-stem were cleaved between the - 1 and the + 1 positions both by Ml RNA and by the reconstituted holoenzyme. These results will be discussed in terms of recognition of the RNase P cleavage site. Finally, variation of the number of base-pairs in the acceptor-stem (or T-stem) resulted in changes in both Michaelis-Menten constants, K, and k,,,.

1019

0

1992 Academic

Press

Limited

8. G. Svdrd and L. A. Kirsebom

1020

2. Materials (a) Plasmid

and Methods constructions

‘The tRNATY’Su3 gene and different mutant derivatives were synthesized together with the phage T7 promoter on an oligo DNA synthesizer and ligated into pUC19 between the &%oRI and Hind111 sites as described elsewhere (Kirsebom $ Sviird. 1992). These gene constructs carry FokI sites downstream from the tRNA genes so that after cleavage with FokI and transcription with T7 DNA dependent RNA polymerase (Milligan et al,; 1987) products of the same sizes as the natural %n viva precursor to tRI\‘ATy’Su3 were generated (Vioque et al., 1988; Kirsebom &i. Altman. 1989). The different gene constructs were verified by DNA sequencing according to Sanger et al. (1977). The construction of the Ml RNA gene behind thr: T7 promoter has been described elsewhere (Vioque et al.. 1988). These plasmids were transformed into E. coli strains HBlQl (Maniatis et aZ., 1982), XLl-Blue (Bullock et al., 1987) or DH5a (Hanahan, 1985) which were made competent according to standard procedures. (b) R.!Vase P assay The RNase P and Ml RNA activities were monitored as previously described (Guerrier-Takada et al.. 1983; Vioque et al., 1988) in standard assay buffers (GuerrierTakada et al.. 1988). The K, and k,,, determinations were done as described by Kirsebom & Altman (1989) and Mirsebom &. Sv%rd (1992).

(c)

Determination

of the cleavage site

The lengths of the different 5’.cleavage products were determined using an RNA ladder prepared as described by Wagner & Nordstrtim (1986). In addition, we compared the sizes of the 5’-fragments derived from rleavage of the various precursors with the 5’.fragments derived from cleavage of a tRNA precursor known to generate two .5-fragments which are 42 and 41 nucleotides long (L. A. Kirsebom. unpublished results). Verification of the sit.e of cleavage was done using the following procedure: the precursors were labelled with [a-32P]GTP, [E~‘P]ATP or [E-~*P~UTP. The choice of which [a-3’P]ETP to use to label a precursor depended on which clea,vage site we wanted to verify. After cleavage with RKase P or Ml RNA the reaction products were separated on an 8% (w/v) polyacrylamide gel in 7 :il-urea TEH-b&leer (90 mM-Tris-borate and (pH 8.5): 2.5 mu-EDTA). The 5’-matured tRNAs were cut out and eluted from the gel using TIC as elution buffer (Maniatis et al., 1982). The so-obtained 5’.matured tRNAs were digested to completion wit’h RNase Tl, RNase T2 and pancreatic RNase A, and the products were analysed by 2-dimensional thin-layer chromatography according to Xishimura (1972) in order to detect the 5’.phosphatecarrying nucleotide. The frequency of cleavage at different positions was quantified from the relative amount of 5’-cleavage products generated from cleavage of the precursors which were clea.oed at more than 1 position. The relative amount of g-cleavage products was determined by densitometry. (d) Stmctural

mapping by RNase Tl, CMCT and R,Vuse H

Unlabelied precursors were labelled at the 3’ end with (5’-32P]pCp according to England et al. (1980) and Guerrier-Takada & Altman (1984). The 3’.labelled

precursor was separated from free [5’-32P]pCp on a G50 spun-column (Maniatis et al., 1982) and purified on an 8% polyacrylamide sequencing gel. Labeiled tRNA precursor was incubated in standard assay buffer (50 m&l-Tris. HCl (pH 7.6), 100 mM-PjH,Cl, 100 mM-MgCi, and 5% (v/v) polyethylene glycol) for 2 min at 37°C and then chilled on ice. Partial RNase 7’1 digestion and analysis of the RNase Tl cleavage products were performed according to Guerrier-Takada & Altman (1984). The tRNA precursors were chemically modified with l-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide methop-toluene sulphonate (CMCTT) as described elsewhere (Mougel et al.; 1987; Shiraishi & Shimura? 1988; Ohman & Wagner, 1989). Each modification mixtIme contained 01 pmol of precursor tRNA together with 0.6 pg of denatured pUC19. Reactions were performed at 37°C and samples were withdrawn after 5 and 30 mm, precipitated with ethanol and subjected to primer-extension according to Stirling et al. (1989) using an oligonucleotide (5’.GGGGAAGGATTCGAACCT-3’) complementary to positions +51 to +68 in the tRNATY’Su3 precursor (see Fig. 1). The primer-extension products were separateld on an 8% polyacrylamide sequencing gel. To determine the positions of modification in the tRKA precursors, WE’ used a sequencing ladder to the t.RXA’%u3 gene construct using the same primer as above. DKA seyuencsing was performed according to Sanger et al. (1977) For the RNase H experiment, 1 pmol of the tRNATY’Su3 precursor and some of its mutant derivatives were incubated at 37°C in standard assay buffer (see above). After 2 min, 2 pmol of the oligonucleotide, 5’.CACGGGGTAATG-3’ (designated oligo-1). complementary to positions - 11 to + 1 in the tRKATY’Su3 precursor (see Fig. 1) was added and the incubation was continued for 1.5 min at 37°C. We also used the oiigsonucleotide 5’.GUGGGGGTAATG-3’ (designated (oligo-IT). complementary to positions - 11 to + I in p&13,-5’SS8 (see Fig. 3). The mixture was then chilled on ice and the assay conditions were adjusted to allow cleavage by RNase H (Donis-Keller, 1979). The mixture cont,ainecl 0.55 unit of RNase H and 0.2 pmol of tRKA precursor hybridized with the oligonucleotide, and cleavage was preformed for 2.5 min at 37°C. The reaction was stopped by, adding an equal volume of 10 w-urea and the reaction l)roducts were separated on an 8% sequencing gel according 1’0 Sanger et al. (1977).

3. Results (a) The selection of the E. coli &Vase f’ cleavage site is not rigidly determined by the lengthy of the acceptor-stem

ami

T-stem

0s a t&VA

precursor

It has been suggested that the locat,ion of t!he site of cleavage in the RNase I’ cleavage reaction is determined by the length of the T-st,em a,nd amino acid acceptor-stem of a tRNA precursor (Carrara et al., 1989; Kahle et al., 1990). Consequently, the site of cleavage would shift if the number of base-pairs in the acceptor-stem and/or T-stem were changed. Therefore, to test this model, we constructed mutant derivatives of the precursor to t~ItNATy’Su3, which carried shortened or ext)ended acceptor-stems and/or T-stems, as outlined in Materials and ___t Abbreviations used: CMCT. 1 -cyclohexyl-3-(2. morpholinoethyl)-carbodiimide metho-p-toluene sulphonate; bp. base-pair(s); EGS, external guide sequence; ES, enzyme-substrate.

Xubstrate Recognition and RNase P Cleavage

QsU3-61

102E

~Su3-XAT

Figure 1. The predicted secondary structures of the precursor to tRPu’ATY’Su3 and the acceptor-stems for some of the different mutant precursors are illustrated. (The D, T and anticodon loops and stems of the different mutant tRKA precursors are not shown.) The wild-type tRNATY’Su3 precursor is designated pSu3 whereas the different mutant precursors are designated as indicated in the Figure. The boxed nucleotides were substituted as indicated. The arrows mdicate the RNase P cleavage sites. The numbering of the tRNA precursor is according to Sprinzl et al. (1991). Methods. Their predicted secondary structures are shown in Figure 1. These mutant tRNA precursors were incubated with Ml RNA or holoenzyme. The sites of cleavage (indicated by arrows in Fig. 1) were determined from the mobilities of the L&cleavage products and verified as outlined in Materials and

No Enzyme added 1m

Methods. The results from these experiments are shown in Figure 2 and summarized in Figure 7. Cleavage of the precursors where the + 16,. + 726 or the - 1U. + 73A base-pair was deleted resulted in 5’cleavage products which were 41 nucleot’ides long both in the absence (Fig. 2, lanes 10 and 12) and

Ml RNA Ml RNA + C5 I I I 1 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Figure 2. Processing in vitro of different precursors by wild-type Ml RSA and reconstituted holoenzyme at 37 “C. ‘I’hc cleavage activities were monitored as outlined in Materials and Methods. The final concentrations of tho reactants were as follows: precursors, 0.06 pmol/pl; Ml R?U’A in the absence of 65, 092 pmol/pl (941 pmol/@ of Ml 1lXA when @3-3X was cleaved) and 00016 pmol/yl in the presence of C5 (0.0082 pmol/@ of Ml RNA when pSu3-RX was cleaved). Tn the absence of C5, the reaction mixture contained 100 mr\l-Mg 2+ The cleavage products and the precursors were separat,ed on an 11% denaturing polyacrylamide gel. Lanes 1 to 7: pSu3, pSu3-40/41, pSu3-A+ lb+ 72, pSu3-A+ IA+ 72A--2; pSu3-AlA+ 73, pSu3-3X and pSu3-61, respectively, no enzyme added; time of incubation (toi) = 120 min; lanes 8 to 15, Ml RNA added; lane 8, pSu3, toi = 15 min; lane 9; pSu3-40/41, toi = 3 min; lane 10, pSu%A+ lA$72, toi = 3 min; lane 11, pSu3-A+lA+72A-2, toi = 30 min; lane 12; pSu3-A-lA+73, toi = 3 min; lane 13, pSu3-3X, toi = 60 min; lane 14: pSu3-3X, toi = 120 min; lane 15, pSu3-61, toi = 3 min; lanes 16 to 22, reconstituted holoenzyme added; Lane 16; toi = 3 min; lane 18, pSu3-A+ lb+ 72, toi = 3 min: lane 19; pSuJpSu3, toi = 5 min; lane 17, pSu3-40/41, A+lA+72A-2, toi = 30 min; lane 20, pSu3-AlA+73, toi = 3 min; lane 21, pSu3-3X, toi = 3 min; lane 22, pSu3-61, toi = 3 min.

1022

8. G. &v&-d and L. A. Kirsebom

-uoAcCCCO -o-* E--C o-c u--A 0-c o-c o-c P-% pSu56

A c u A c A

-mJAcccc(I -0-A

-ml~CccCD --to

o-c o-c xl--A G-C o-c o-c P-1

0-c (i-c 0-f. o-c 0-c G-C No-% p&3-A74

c

A-O

* c D A c c

A

-vmccccc -II-* o-c o-c O--A o-c G-C 0-c P-T ps3+2c)

A c cl A c c fl

A c

-mmxcco -WV

psu3-ss7

A c 0 A c c A

-m*ccccc +A G-C 0-c 0-L 0-c (i--c 0--c ,0-c

\

psu3-sss7

c tJ c 0 o-c 0-A G-C o-c o-c /G-c\

psu3-ssu

A c

-:

: : : G-C G-C c-c P-k psu3-SSIO

A c tt

i

-“=~E c o-c u-* G-C o-c G-C ,0-c

E c

\

ps3-S’SSS

n

ii -uo*cccc

c- G

“g-l li-?I 0-c o-c 0-c p-c p.wl-syc art.3 psl%syc

o-c

\

1

Figure 3. The predicted secondary structures of the precursor to tRNATY’Su3 and the acceptor-stems for some of the different mutant precursors which harboured a reduced number of base-pairs in the acceptor-stem are illustrated. (The D, T and anticodon loops and stems of the different mutant tRNA precursors are not shown.) The wild-type tRNATy’Su3 precursor is designated pSu3 whereas the different mutant precursors are designated as indicated in the Figure. The boxed nucleotides were substituted as indicated (A, deletion). The arrows indicate the RNase I’ cleavage sites. The numbering of the tRPjA precursor is according to Sprinzl et al. (1991). presence of C5 (Fig. 2, lanes 18 and 20). These results suggest that cleavage occurred between positions - 3 and -2 in both these mutant tRNA precursors generating 5’-matured tRNATYTSu3 derivatives with eight base-pair (bp) acceptor-stems (the numbering in this paper refers to that given for the wild-type tRNATY’Su3 precursor, see Fig. 1). Similarly, cleavage of pSuS-40141 (which carried an insertion of a G. C base-pair in the acceptor-stem, see Fig. 1) both in the absence (lane 9) and in the presence of C5 (lane 17) resulted in 5’.matured tRNAs with 8 bp acceptor-stems. This suggested that cleavage occurred between the - 1 and the + 1 positions (see Fig. 1). Taken together, these results suggest that the selection of the cleavage site is not merely determined by the length of the acceptorstem and T-stem. However, as a result of RNase P cleavage, two naturally occurring tRNAs have 8 bp acceptor-stems, tRNAHis and tRNASeCys (Orellana et al., 1986; Burkard & &ill, 1988). To investigate of the length of the acceptorfurther the importance stem with respect to the position of cleavage we studied cleavage of a tRNATY’Su3 precursor derivative (p&3-3X) which carried an insertion of 3 bp (one G. C and 2 U. A bp) in its acceptor-stem (see Fig. 1). This precursor was cleaved with a significantly reduced efficiency both by Ml RNA and by the holoenzyme. The mobilities of the 5’-cleavage

products derived from cleavage of the wild-type and this mutant precursor were the same whether we used Ml RNA or holoenzyme (Fig. 2, compare lanes 8 and 16 with lanes 13, 14 and 21). This suggests that pSu3-3X was cleaved between positions - 1 and + 1 generating a 5’.mature tRNA. with a 10 bp acceptor-stem. However, we also observed some residual cleavage downstream from position -t 1 and verification of the cleavage sites as outlined in Materials and Methods demonstrated the presence of both pGp and pup at the 5’-end after cleavage with Ml RNA and with the holoenzyme (data not shown). In conclusion, cleavage occurred mainly at position + 1 (as indicated in Fig. 1) in spite an extension of the acceptor-stem with 3 bp. We also extended the length of the T-stem of the wild-type and the mutant tRNATY’Su3-40141 precursor with one G. C base-pair. The resulting precursors were designated p&3-61 and pSu3-XAT, respectively (see Fig. I). Both these precursors were cleaved at the same position as the wild-type precursor as determined from the mob&ties of the $-cleavage products generating 5’-matured tRNAs with 7 and 8 bp long acceptor-stems, respectively (pSu3-61: Fig. 2, lanes 15 and 22; p&3-XAT: Fig. 6(a), lanes 10 and 15). From these results, we conclude that the location of the E. coZi RNase P cleavage site is not dependent in a simple way on the length
Recognition

Xubstrate

and RNase

P Cleavage

amino acid acceptor-stem and/or the T-stem of the tRNATy’Su3 precursor. The pSu3 - A + 1A + 726 - 2 mutant will be discussed below.

-8 lip;.

(b) RNase P cleavage and base-pairing

near

1023

RNaseH

34’

-10 5ii

-sss

-SSlO

77

95

11

-+-+-+-+-+

the site of cleavage

(i) Structural which

analysis base-pairing

of tRNATy’Xu3 near

the cleavage

precursors

in site has been

disrupted Previous studies have shown that the amino acid acceptor-stem of a tRNA precursor is important for the cleavage reaction (Burkard et al., 1988; Green & Vold, 1988; Guerrier-Takada et al., 1989; Holm & Krupp, 1992; Kirsebom & Svard, 1992). Thus, in an attempt to investigate further the significance of the acceptor-stem we decided to reduce the number of base-pairs near the cleavage site in the tRNATY’Su3 precursor. Details about the construction of these mutant tRNA genes are given in Materials and Methods. As shown in Figure 3 these tRNATY’Su3 precursor derivatives carried either deletions at the 3’.end or base substitutions at positions +70 to + 74 ((ACCAC-) or at positions - 2 to + 1 (-GUG-). The precursors to tRNATY’Su3-8 (pSu3-8), tRNATYTSu3-10 (pSu3-IO), tRNATY’Su3-SS8 (pSu3tRNATY’Su3-SSlO and (psu3-ss10) fw, tRNATY’Su3-5’SS8 (pSu3-5’SSS) are potentially single-stranded on both sides of the cleavage site. These precursors (except pSu3-5’SSS) were subjected to partial digestion with RNase Tl (see Materials and Methods) to probe their structures, in particular near the cleavage site. The results from this experiment showed that pSuY-10 and pSu3SSlO were sensitive to RNase Tl at positions downstream from the RNase P cleavage site. We also observed very weak RNase Tl cleavage sensitivity near the RNase P cleavage site in pSu3-8 whereas pSu3 was not cleaved by RNase Tl in this part of the precursor (data not shown). In additional experiments, wild-type and mutant precursors (except pSu35’SS8) were modified with CMCT. This reagent reacts with single-stranded U and to a lesser extent with single-stranded G (Ehresmann et al., 1987). Thus, if the secondary structures predicted in Figure 3 were correct then the - lU, in pSu3-8 and pSu3SS8 and - 1U and + 3U in pSu3-10 and pSu3SSIO would be sensitive to modification by this reagent. The results showed that the - 1U in pSu3-8 and both - 1U and + 3U in pSu3-10, pSu3-SS8 and pSu3-SSlO were accessible to CMCT (data not shown). Finally, we mixed the wild-type and mutant tRNA precursors (pSu3-8, pSu3-10, pSu3SSB and pSu3-SSlO) with a deoxy-oligonucleotide (oligo I) complementary to positions - 11 to + 1 in pSu3 (see Fig. 3) as described in Materials and Methods. Addition of RNase H resulted in cleavage of the mutant precursors, whereas the wild-type precursor was cleaved very poorly (Fig. 4). Similarly, pSu3-5’SS7 and pSu3-5SS8 were also cleaved by RNase H after addition of a deoxynucleotide (oligo II) complementary to positions -11 to + 1 in pSu3-5’SS8 (see Fig. 3) (data not

Figure 4. Probing of the structures of some of the different mutant derivatives of tRNATY’Su3 by RNase H cleavage. The experiments were performed as outlined in Materials and Methods. Samples were loaded onto an 8% polyacrylamide sequencing gel. The minus and plus signs indicate the absence and presence of RNase H; respectively. Lane 1, pSu3 no RNase H added; lane 2, pSu3 RNase H added; lane 3, p&13-8 no RNase H added: lane 4, pSu3-8 RNase H added; lane 5, pSu3-10 no RNase H added; lane 6, pSu3-10 RNase H added; lane 7; p&13-SS8 no RNase H added; lane 8, pSu3-SS8 RNase H added; lane 9, pSu3-SSlO no RNase H added; lane 10; pSu3-SSlO RNase H added; lane 11; pSu3 cleaved for 15 min at 37°C with Ml RNA in the absence of C5.

shown). From these results we conclude that pSu3-8, pSu3-10, pSu3SS8, pSu3-SSlO and pSu35’SSB are single-stranded on both sides of the site of cleavage, as indicated in Figure 3. The results of partial RNase Tl digestion and of CMCT modification of the various mutant precursors did not reveal any significant changes in the structure in other regions when compared to the wild-type precursor. (ii) Base-pairing prerequisite

for

near the site of cleavage cleavage

is not a

The precursors discussed in the previous section were analysed and found to be cleaved by Ml RNA in the absence or presence of C5, although two of the precursors carried only four base-pairs in their accept,or stems. The results are shown in Figure 5 and the cleavage sites are summarized in Figure 7. (The kinetics of cleavage are discussed below). The 5’-cleavage product (Fig. 5(a), lane 8) of pSu3-10 produced by Ml RNA is represented by three bands, a major band (A) which migrated at the same position as the 5’-cleavage product derived from the wild-type precursor, and two minor bands (B and 6) which migrated slightly faster than the major band. Analysis of fragments B and C, as outlined in Materials and Methods, showed that they were 41 and 38 nucleotides in length, respectively. This suggested that cleavage of pSu3-18 by Ml RNA occurred at three positions as indicated in

S. G. Svtird and L. A. Kirsebom

Figure 3; position + 1, generating the major band, A, in addition to positions - 2 and --5 generating the minor bands, B and C. In contrast, in the presence of the C5 protein, cleavage occurred only at the + 1 position. The sites of cleavage were further characterized as outlined in Materials and Methods (data not shown). The precursors p&13-6 and p&13-8 were cleaved at only one position! + 1, both by Ml RNA and by the holoenzyme (Fig. 5(a), lanes 6, 7, 10 and 11). The cleavage pattern for p&13-6 is in agreement with Guerrier-Takada et al. (1989). We also observed cleavage of precursors to tRNATY’Su3, where 12 and 14 nucleotides were deleted from the 3’.end. However, in these cases cleavage occurred at other positions than at the + 1 position with a significantly reduced rat,e (data not shown). In conclusion, these results demonstrate that cleavage by E. coli RNase P or its catalytic subunit Ml RNA is not dependent on base-pairing at or near the site of cleavage. However, cleavage requires at least four base-pairs in the acceptor-stem of the tRNATY’Su3 precursor. Furt)hermore, in agreement with previous reports (Green 8~ Vold, 1988; Guerrier-Takada et al., 1989), these results demonstrate that CCA is not required for cleavage. This is in contrast to what has been observed using a model substrate (McClain et al., 1987). In addition, our results show that cleavage by %!I RNA or RNase P is not dependent on the other CCA sequences in the tRNATY’Su3 precursor (see Fig. 3). The mutant tRNATYTSu3 precursors which carry six (pSu3SS8) or four (p&3-SSlO) base-pairs in the acceptor-stem as a result of base substitutions were cleaved at more than one position, + 1 and -2, in the absence of C5 (Fig. 5(b) and (c), lanes 9 and 10, respectively). The 5’.fragment derived from cleavage at position -2 is of the same size ats band B in Figure 5(a) (data not shown). The sites of cleavage are indicated in Figure 3. The frequency of cleavage at the - 2 position by Ml RNA alone was significantly increased relative to cleavage of pSu310 at this position (see Fig. 7). We did not detect any pGp after cleavage with Ml RNA in the -

Figure 5. Processing in vitro of different precursors by -wild-type Ml RNA and reconstituted holoenzyme at) 37°C. The cleavage activities were monitored as outlined in Materials and IMethods. The final concentrations of the reactants were as follows: precursors, 0.053 pmol/pl; Ml R,hiA in the absence of C5, 0.02 pmol/,nl and 0.0016 to 08024 pmol/nl in the presence of C5. In the absence of C5, the reaction mixture contained 100 mivr-Mg2+. The cleavage products and the precursors were separated on a 105% or 125% denaturing polyacrylamide gel. (a) Lanes 1 to 4, pSu3, pSu3-6, pSu3-8 and pSu3-10, respectively, no enzyme added, time of incubation (toi) = 30 min; lanes 5 to 8: MJ RSA added; Iane 5, pSu3, toi = 15 min; lane 6, p&S-6, toi = 3 min; lane 7, pSu3-8, toi = 3 min; lane 8, p&13-10. toi = 30 min; lanes 9 to 12, reconstituted holoenzyme added; lane 9, p&3. toi = 5 min; lane 10, pSu3-6,

toi = 3 min; lane 11, pSu3-8, toi = 3 min; lane 12, pSu310, toi = 30 min. (b) Lanes 1 to 5, p&13. pSuY-A74; pSu3SS7, pSu3SS8 and pSu3-SSJO, respectively, no enzyme added, toi = 45 min; lanes 6 to 10, MB RNA added; lane 6, pSu3, toi = 15 min; lane 7. pSu3-A74, toi = 3 min; lane 8, pSu3SS7, toi = 3 min; lane 9. pSu3SS8, toi = 10 min; lane 10, pSu3-SSlO, toi = 30 mm; lanes 11 to 15, reconstituted holoenzyme added; lane 11; pSu3, toi = 5 min; lane 12, pSu3-A74, toi = 3 min; lane 13, pSu3SS7, toi = 3 min; lane 14, p&13-$S8, toi = 12 min; lane 15, pSu3-SSlO, toi = 45 min. (c) Overexposure of the 5’-cleavage products in (b). (d) Lanes J to 4, pSu3-5’S%‘, pSu35SS8, pSu3-SyC and pSu3SyG. respectively. no enzyme added, toi = 40 min; lanes 5 to 8, MI RNA added; lane 5, pSu3-5’SS7, toi = 17 nun; lane 6. pSu3-5’S%, toi = 30 min; lane 7, pSu3-SyC, toi = 30 min; lane 8, toi = 30 min; lanes 9 to 12, reconstituted pSu3SyG, holoenzyme added; lane 9, p&3-5’SS7, toi = 5 mm; lane 10, pSu3-5’SS8, toi = 30 mm; lane 1 I, p&3-SyC, toi = 30 min; lane 12, pSu3-SyG, toi = 41 min.

Substrate

Recognition

and RNase

P Cleavage

1025

Figure 6. Cleavage of wild-type and some mutant tRIGATY’Su3 precursors in which the nucleotide at the + 1 position was substituted as indicated in Figs 1 and 3. (A) Cleavage of pSu3-40/41 derivatives (and pSu3-XAT). by Ml RNA in the absence and presence of C5. (B) Cleavage of + 1 derivatives of pSu3-10 by Ml RKA in the absence of C5. The experiment was performed at 37°C as outlined in Materials and Methods and in the legend to Fig. 2. (A) Lanes 1 to 5, pSu3-40/41. pSu3-40/41+ lA, pSu3-40/41+ lC, pSu3-40/41+ 1U and pSu3-XAT, respectively, no enzyme added, time of incubation (toi) = 10 min; lanes 6 to 10, MI RNA added; lane 6, pSu3-40/41, toi = 10 min; lane 7, pSu3-40/41+ IA, toi = 10 min; lane 8, pSu3-40/41+ 1C. toi = 10 min; lane 9, pSu3-40/41 f 1U, toi = 10 min; lane 10; pSu3-XAT, toi = 15; lanes 1I to 1.5, reconstituted holoenzyme added; lane 11, pSu3-40/41, toi = 5 min; lane 12, p&&40/41 + IA. toi = 5 min; la,ne 13: pSu340/o/41+ IC, toi = 5 min; lane 14, pSu3-40/41+ lU, toi = 5 min; lane 15; pSu3-XAT, toi = 7 min. (B) Lanes 1 to 6, p&1310, p&3-lOA+ 1, pSu3-lO+ lU, p&3-+ 1A and pSuY-10+ 1C respectively, no enzyme added, toi = 42 min for p&13-10; and 82 min for the + 1 mutant derivatives; lanes 6 to 10, Ml RNA a.dded; lane 6, pSu3-10, t.oi = 36 min; lane 7, pSuYlOA+ 1, toi = 75 min; lane 8, A ~Su3-lO+ 1U. toi = 75 min; lane 9: p&13-10+ IA, toi = 75 min; lane 10, pSu3-IOf 1C. t.oi = 75 min.

absence of @5 using [a32P]UTP-labelled @US-SS8 or pSu3-SSlO (data not shown). This result suggested that cleavage of these two mutant precursors at the - 2 position resulted in a product which was cleaved a second time, presumably at position -t- 1. The holoenzyme cleaved pSu3-SS8 and pSu3-SSlO at position + 1 (lanes 14 and 15) with some residual cleavage at position -2 (data not shown for pSu3SE%). In addition, cleavage of pSu3-SSlO occurred at an additional position in the absence of C5 (indicated by X in Fig. 5(b), lane 10: the 5’kleavage product was visible on the original autoradiogram). In contrast, base substitutions of the f73A and/or the $74C, as in pSu3-SS7 and pSu3-A74 (see Fig. 3) resulted in cleavage only at the + 1 position both in the absence and presence of C5 (Fig. 5(b), lanes 6 and 11). We also disrupted base-pairing at and proximal to the cleavage site by substituting the nucleotides -2: - 1 and +I with C, A and C (see at positions Fig. 3). This resulted in aberrant cleavage only when the nucleotides at all three positions were changed simultaneously as in the pSu3-5’S% derivative. This was observed both in the absence and presence of C5 (Fig. 5(d), lanes 6 and 10; see Fig. 7). Nevertheless, cleavage occurred mainly between positions - 1 and + 1. One of the cleavage sites on pSu3-5’S% for the holoenzyme was at a position upstream from - 2. We did not observe any 5’-matured tRNA product as a result of cleavage at this position, suggesting that the cleavage product was cut again, presumably at position -I- 1. The 5’-

cleavage product derived from cleavage of pSu3 and the major 5’kleavage product generated in cleavage of pSu3-5’S% were shown to be of the same size (data not shown). Substitutions of the - 1U and the -2G (pSu3-Fi’SS7) or the - 2G (pSu3-( - ZC)) resulted in cleavage only at the + 1 position (Fig. 5(d), lanes 5 and 9, data not shown for psu3-( -2C)). (iii) Changing cleavage

the nucleotides proximal to the site without disruption of base-paising

Cleavage at more than one position might be the result of the absence of base-pairing at and near the site of cleavage. Thus, we constructed mutant tRNATY’Su3 precursors in which the base-pairing in the acceptor-stem was intact but the nucleokles at -2, -1, fl, +72, +73 and +-74 were positions altered compared to the wild-type precursor. These mutant precursors were designated pSu3-SyC and pSn3-SyG (see Fig. 3). Both in the absence and presence of C5 these two mutant precursors were cleaved at positions + 1 and -2 as indicated in Figure 3. The results are shown in Figure 5(d) (lanes 7; 8; lid and 12) and Figure 7. The pSu3-SyG precursor was also cleaved with a low frequency -2 and - 1 both by 31111RNA between positions and by the holoenzyme. The observed differences in mobility of the 5’-matured tRNAs are attribated to differences in their primary structures since residual RNA secondary structure has previously been observed in denaturing polyacrylamide gels @egg & Thurlow, 1990).

X. G. Sviird

1026

Precursor

-2c

and L. A. Kirsebom

5'SS7

5'SS8

A-lA+73

+lA 40'41

f%41

40/41 +1c

A+l.A+72

3x##

-10

-10 flu

-10 +1c

Precursor

Precursor

40/41

XAT

Figure 7. Summary of the cleavage site of the tRNA precursors used in this study. The sites of cleavage were determined as outlined in the text. Kumbers given in parentheses represent the percentage of eleavage at the different positions and were calculated as outlined in Materials and Methods. (*), Some residual cleavage was observed at position - 2 but the percentage of cleavage was not determined; (**), cleavage upstream from the -2 position was observed but the exact position was not mapped; (# ), cleavage occurred either at the -4 or - 5 position; (# # ), the p&3-3X was cleaved at a 2nd position most likely at position + 4, see the text; (y), the pSu3-SyG was cleaved at the - I. position with a iow frequency both in the absence and presence of C5. The numbering refers to the numbering given for pSu3 = wild-type, see Figs 1 and 3. ND, not determined.

Taken together, these data show that the absence or presence of base-pairing at and proximal to the site of cleavage in pSu3 resulted in cleavage at more than one position. This suggests, therefore, that it is not base-pairing which is important for the selection of the cleavage site but rather the identity of the nucleotides at and near the site of cleavage. This is consistent with the finding that the truncated precursors were cleaved mainly between the - 1 and the + 1 positions. Moreover, it has previously been reported that a mutant Ml RNA cleaves a truncated tRNATY’Su3 precursor at multiple sites upstream from the f 1 position (Guerrier-Takada et al., 1989). Thus, together with the results reported here we can conclude that both wild-type and mutant Ml RNA can cleave (depending on the substrat,e) at positions other than the + 1 position. This is in keeping with a previous report (Krupp et al.; 1991). (iv) The importance of the identity of the nucleotide at position + 1 in the cleavage reaction As shown tions -2, occurred at presence) of identity of

above, when the nucleotides at posi- 1 and + 1 were altered, cleavage several positions in the absence (or C5. Earlier reports have shown that the the nucleotide at the cleavage sibe is

crucial in the selection of the cleavage site on the et al.. 1988; Green & tRNAHi” precursor (Burkard Void, 1988; Kirsebom & Xv&l; 1992). Thus, we argued that correct cleavage of the tRNJ4TY’Su3 precursor might be dependent on the identity of the nucleotide at the cleavage site. To test this we substituted the + 1G in the wild-type tRN.ATY’Su3 precursor (pSu3) as indicated in Fjgure 1. In addition, we changed (or deleted) the nucleotide at the cleavage site in some mutant precursors, pSu3-401 41, pSu3-10, pSu3-SS8 and pSu3-A + ld + 72, see Figures 1 and 3. The .tRNATY’Su3 precursor was cleaved at the normal site in the absence and presence of C5 independent of the identity of the nucleotide at the + 1 position (data not shown). In contrast, changing the nucleotide at the sites of cleavage in the mutant precursors resulted in significant cleavage at other positions. These results are shown in Figures 2 and 6, and summarized in Figure 7. The cleavage sites were inferred from the mobilities of the 5’.cleavage products. A pyrimidine at the + 1 position in pSu340/41 resulted in a shift of the cleavage site to position +2 in the absence or presence of C5 (Fig. 6(a), lanes 8, 9, 13 and 14). In contrast, a G t)o A substitution did not result in a change of the cleavage site (lanes 7 and 12). When we replaced the

Substrate

Recognition

+ 1G with any of the other nucleotides in the truncated precursor pSu3-10 we observed significant levels of cleavage at several positions upstream from the normal cleavage site in the absence of C5 (Fig. 6(b)). Similarly, when the guanosine at the cleavage position in pSu3-A+ lAf72 was deleted (see Fig. l), cleavage by Ml RNA alone occurred at more than one location (Fig. 2, lane 11). We noted that this latter precursor was cleaved at only one position in the presence of C5 (Fig. 2, lane 19). The combined data indicate that the identity of the nucleotide at the cleavage site is important for the location of the cleavage site under certain conditions. However, the G at the + 1 position in the wild-type tRNATy’Su3 precursor is not esssential for cleavage at this position. Changing the nucleotide at position + 1 in pSu3-8 (see Fig. 1) did not alter the location of the cleavage site (data not shown). (c) Variation

of the number

of base-pairs

in the

acceptor-stem or T-stem results in changes in both kinetic constants under steady-state conditions

We also determined the kinetic constants under steady-state conditions for the wild-type and selected mutant precursors. The experiments were done using both Ml RNA and holoenzyme as outlined in Materials and Methods. The results are shown in Table 1. We observed a significant increase in K, as a result of deletion of 6, 8 or 10 nucleotides from the 3’ end of the tRNATY’Su3 precursor in the absence or presence of C5 (we consider a factor of more than 2 as significant). An enhancement in K,,, was also observed when the base-pairing was disrupted by base-substitutions at positions near the cleavage site (pSu3-SS8, pSuSSS10, pSu3-5’SS8 as well as pSu3-A+ lA+72). The latter precursor carried a deletion of the + 1G. +72C base-pair (see Fig. 1).

Michael&Menten

constants

of the Ml

RNA

and RNase

P Cleavage

Cleavage of pSu3-5’SS8 and pSu3-A+ 1 resulted in an increase in K, only in the absence of C5. These results are in agreement with the finding that the primary structure and length of the acceptor-stem are of importance for an efficient interaction with the enzyme (Kirsebom & Sviird, 1992). Substitutions of the nucleotides at positions -2, - 1, + 1, + 72, +73 and +74 without disruption of the base-pairing (pSu3-SyC) also resulted in an impairment in the interaction between the precursor and Ml RNA in the absence of C5 as determined by the K,,, value. In contrast, addition of C5 to the cleavage reaction resulted in no difference in the K, values for cleavage of pSu3 and pSu3-SyC. These results suggest that the identity of the nucleotides near the site of cleavage, rather than their potential to form base-pairs, are also important for the enzyme-substrate interaction, in particular in the absence of C5. Surprisingly, pSu3, pSu3A74, pSu3-( -2C) and pSu3-5SS7, were processed with the same K, both by Ml RNA and reconstituted holoenzyme. We also noted that addition of C5 resulted in a change in K, values in a substratedependent ma.nner, in agreement with previous reports (Peek-Miller & Altman, 1991; Kirsebom & Svard; 1992). In comparison to cleavage of the wild-type precursor, disruption of one to three base-pairs either by a deletion or by introducing base-substitutions, resulted in a significant increase in k,,, in the absence of C5. Addition of C5 resulted in an increase which carried in kc,, only for those precursors changes in the 3’-proximal part’ of the aeceptorstem. Disruption of five base-pairs in the aceeptorstem (pSu3-10 and p&3-SSlO) or deletion of the + 1G. -+ 72C base-pair (pSu3-A -t 1A + 72) resulted in relative to cleavage of a kc,, which was unchanged the wild-type precursor using Ml RNA. Similarly, cleavage by the holoenzyme of pSu3-SSlO, pSu3

Table I and RNase P cleavage reaction in this study

Ml RNA

-~ Substrate

k C(lf

pSu3 pSu3-6 pRu3-8 pSu3-10 p&13-A74 psu3-SS8 psua-SSlO psu3-( -2C) psu3-(5’SS7) psu3-(5’SS8) psua-syc p&13-A + 1A + 72 pSu3-61

0,22 35 5.8 0.25 2.0 2.1 0.34 0.83 1.7 048 0.13 0.21 1.4

2..5 17 143 11.4 26 9.8 7.8 28 1.3 9.3 13 65 94

1023

of some mutant

substrates

used

Ml RNA+65

C8 2@6 406 2.2 769 21.4 44 296 131 95 1.0 3.2 15

104 1%7 42 1.9 37.4 44.4 lo.3 11.2 il.2 2.3 1.2 2.9 24.2

1.1 7.2 l3.5 10.5 1.5 5.3 13.9 1.6 1.8 1.5 2.1 1.0 46

991

260 311 18.1 2493 838 74.3 700 622 153 57 290 526

The k,,,, and K, values given are average values from several independent experiments with experimental errors of i 32 yO (based on 9 independent experiments) and +44% (based on 8 independent experiments), respectively, using pSu3 and wild-type h&enzyme.

I.028

S. G. Xv&d

and L. A. Kirsebom

( - 2C) a,nd pSu3-5’SS7 resulted in no change in iE,,,, whereas a decrease in k,,, was observed for pSu3-10, pSu3-5’SSB and pSu3-A + 1A+ 72. The pSu3-SyC precursor was cleaved with approximately the same k cat as pSu3 in the reaction with the Ml RNA alone, whereas in the presence of C5 a reduced k,,t was observed. The k,,,lK, value showed that the cleavage reaction in the presence of C5 was stimulated by the presence of nucleotides on the 3’ side of the acceptor-stem. We also noted that pSu3-A74 was processed more efficiently than the wild-type precursor although it carried a mutation in the CGA. Finally, an extention of the T-stem with one G. C base-pair (see Fig. 1) yielded an increase in both kinetic constants. (These results will be further discussed below.)

4. Discussion (a) Several regions determine

the location

of a wild-type tRNA precursor the RNase P cleavage site

qf

(i j Effects of acceptor-stem length the RNase P cleavage site

on the location

does not depend in a simple manner on the lengths of the acceptor-stem and T-stem. The precursor which carried three extra basepairs in the acceptor-stem (pSu3-3X) was cleaved mainly at the + 1 position. We also observed some residual cleavage at an additional position downstream from the main site of cleavage. Our unpublished observations suggest that the second cleavage site on pSu3-3X was at a position located three base-pairs downstream from the + 1 position as indicated in Figure 1. Previous observations suggest that cleavage of certain tRNA precursors yield products which can be used as substrates for RNase P (Burkard et al., 1988; Kirsebom $ Svard, 1992). Thus, it is also possible that elea,vage of p&$3X at position + 1 generates a product which is subsequently used as a substrate and cleaved at the second position. This results in 5’.Imatured tRNAs with 7 bp acceptor-stems in both cases. In conclusion, it appears that the length of the acceptor-stem plays some role in the location of the cleavage site.

of

In this paper we have investigated cleavage by E. coli RNase P as a function of the number of basepairs in the acceptor-stem and/or T-stem of a natural tRXA precursor, the tRNATY’Su3 precursor demonstrate that mutant (@US). The results precursors which carry varying numbers of basepairs in these stem structures were cleaved by Ml RNA in the absence or presence of C5. Surprisingly, we found that even those precursors with only four base-pairs in the acceptor-stem were cleaved both by MI RNA and by holoenzyme. However, the kinetics of cleavage differed for the different precursors. It has been suggested that the location of the RNase k cleavage site in a tRNA precursor is determined by the length of the acceptor-stem and T-stem (Carrara et al., 1989; Kahle et al., 1990). Thus, the site of cleavage would shift accordingly if the length of the acceptor-stem and/or T-stem were shortened or extended. However, as shown here, extensions of the acceptor-stem and/or T-stem of pSu3 resulted in precursors which were still cleaved between positions - 1 and + 1. In one case RNase P cleavage generated a 5’.matured tRNA with a 10 bp long acceptor-stem (see Fig. 1). Furthermore, a truncated precursor which carried only four basepairs in the acceptor-stem (pSu3-10) was also cleaved mainly between the - 1 and the + 1 positions (see Fig. 3). In contrast, when the - lU’+ 73A or the + IG. + 726 base-pair was deleted cleavage occurred between positions - 3 and -2 generating 5’-matured tRNAs with 8 bp acceptor-stems (see Fig. I). In addition, others have demonstrated that cleavage of the precursors tRNA”‘” and tRNASeCyS by eubacterial RNase P yields 8 bp amino acid acceptor-stems (Orellana et al., 1986; Burkard & Siill, 1988; Holm & Krupp, 1992; Kirsebom & Svard, 1992). From these data we conclude that the location of the cleavage site by the E. coli RNase P

(ii) The location of the cleavage site on the tRlNATy’Su3 precursor ix dependdent on the identity of the nucleotides proximal to the scissile bond The 3’.proximal sequence of the acceptor-stem of a tRNTA precursor has been proposed to function as an external guide sequence (ECS) (Forster & Altman, 1990). Cleavage by MI RNA of the truncated precursor pSu3-10, which carries only four base-pairs in the acceptor-stem, occurred mainly at position + 1 (this study). Our results suggest, therefore, that part of the EGS in pSu3 is not required for cleavage at the normal position. We emphasize that this refers to normal tRNA precursors and not to model substrates. One possibility is that the unpaired nucleotides in pSu3-16 st,ack on the remaining acceptor-stem, which results in cleavage mainly at position + 1. This would also explain why the truncated precursor pSu3-8 was cleaved only at the + 1 position. In a full-length tRNATY’Su3 precursor the nucleotides at positions 72 to 74 can be considered to be part of an extended EGS. Changing these nucleotides as in the precursors pSu3SS8 and pSuSSS10, resulted in cleavage both at, positions -2 and +l (see Fig. 3). In contrast, substitution of the +73A and +74C (pSu3-SS7) or the +74C (pSu3-A74) did not result in aberrant cleavage. In addition, a tRNATY’Su3 precursor which carries a base substitution at position + 72 or a deletion of the +73C is cleaved at’ the same position as the wild-type precursor b’oth in the absence and presence of C5 (LA. results). Taken together, these results suggest that the aberrant cleavage of pSu3-SSIO and pSu3-SS8 is the result of changing the nucleotides at positions +72 to +74 simultaneously and not just by the absence of base-pairing. This is consistent with previous observations that nucleotide substitutions at the Y’-end of the acceptor-stem of a tRNA precursor (at or above the site of cleavage) resulted

Substrate

Recognition

in aberrant cleavage (Burkard et al., 1988; Krupp et al., 1991). In conclusion, we propose that the nucleotides at positions +72 to +74 constitute one domain, within EGS, which is important in the location of the RNase P cleavage site. However, the presence of these bases is not absolutely required for cleavage between the - 1 and the + 1 positions, at least not in the tRNATY’Su3 precursor. Disruption of the base-pairs at and proximal to the site of cleavage by changing the nucleotides at -2, - I and + 1 (pSu3-5’SS8) also resulted in cleavage at several positions but mainly at the + I position (see Fig. 3). However, in t,his case cleavage at the alternative positions was less frequent than observed using pSu3-SS8 and pSu3-SSIO. We know from the results reported here and from our unpublished observations that the cleavage site is not changed as a result of base substitutions at either of these positions. Therefore, the low frequency of aberrant cleavage of pSu3-5’SSS is most likely due to the simultaneous change of the nucleotides at these three positions. We also showed that a G at the cleavage site was crucial in t.he selection of the cleavage site on some mutant tRNA precursors in the absence of 05. In contrast, cleavage between positions - 1 and + 1 of the wild-type tRNATY’Su3 precursor was independent of the nucleotide at the + 1 position. Thus, the significance of the identity of the nucleotide at the cleavage site became evident when other landmarks on the tRNATY’Su3 precursor were deleted or changed. Previous reports have demonstrated the importance of a C at the site of cleavage in the location of the cleavage site on the tRNAHi” precursor or on a chimeric tRNA precursor (Burkard et al., 1988; Green &, Vold, 1988; Kirsebom & Svard, 1992). From the combined data we propose that the nucleotides at positions -2, - 1 and + I constitute a second domain of the tRNATY’Su3 precursor which is important in t.he location of the cleavage site. Within this domain we propose t’hat the + 1G functions as a guiding nucleotide. This might be one explanation for why most tRNAs in E. co& carry a G at the cleavage site (Sprinzl et al., 1991). The importance of a guanosine immediately 3’ to the cleavage-ligation site has also been demonstrated for another ribozyme (Chowrira et al., ISSl). Base substitutions either at positions -2 and - 1 or $73 and +74, as in pSu3-tj’SS7 and pSu3-SS7, respectively, did not result in aberrant cleavage. However, pSu3-SyG, in which base-pairing was restored but the nucleotides at positions - 2, - I, +73 and +74 were altered compared to the wildtype precursor, was cleaved at the -2 and + 1 positions (see Figs 3 and 7). Thus, changes in one domain resulted in cleavage only between the - 1 and +l positions but changes in both domains resulted in cleavage at more than one position. In conclusion, a combination of several regions of a wild-type tRNA precursor determines the selection of the site of cleavage for E. co& RNase P. Among these are the nucleotides at positions -2 to + 1 and + 72 to + 74 in the case of p&3. The length

and RNase

P Cleavage

iO29

of the acceptor-stem appears also to play some role in the location of the cleavage site, as discussed above. Recently, it was shown that the tRNAryrSu3 precursor was cleaved specifically at the -2 position by Mg2+ suggesting that this region of the precursor is involved in Mg’+ -binding (Kazakov & Altman, 1991). Furthermore, the 2’.hydroxyl groups at positions -2, -1, +I and -t-33 (the latter corresponding to position +74 in a tRNA precursor) are important for cleavage of model substrates most likely through the binding of a Mg ion (Perreault & Altman, 1992). Thus, changing the nucleotides in this part of pSu3 might result in a change in Mg 2+-binding. The nucleotides at pasitions -2, -1, +l, -t-72, +73 and +74in pSu3 are of significance in the location of the cleavage site, as we have shown in this study. It, is, therefore, binding near the cleavage conceivable that Mg2+ site is dependent not only on the 2’.hydroxyl groups but also on the identity of the nucleotides at these positions. (b) The kinetics of cleavage and the amino acid acceptor-stem (i) Cleavage

appears

to be substrate-dependent

The mutant precursor to tRNATY’Su3-874. which harbours a C to A change in CCA (the changed C is underlined), was a better substrate than the wildtype precursor. This is a consequence of an increase in k,,, (Table 1). In contrast, an increase in M, results from mutations in CCA in a precursor to theNAn”” (Green 8~ Vold, 1988). Also, the CGA change in a model substrate for RNase P resulted in no detectable cleavage either by Ml RNA or the holoenzyme (M&lain et al., 1987). Thus, the structural changes at the 3’.end of a tRNA precursor is not as dramatic as the structural changes at the 3’end of a model substrate. This might suggest that the D-stem, D-loop: anticodon st,em and antieodon loop help to anchor the precursor on the enzyme. Tn conclusion, different precursors are processed differently in vitro by Ml RNA and RNase P with respect to their rates of cleavage. This supports previous findings where it has been shown both irk viva and in vitro that cleavage by RNase P is substratedependent (Kirsebom et al., 1988; Kirsebom & Svard, 1992; Gurrier-Takada & Altman, 1992). An “induced fit” model could explain these observations, as discussed by Guerrier-Takada et al. (I. (ii) The importance of base-pairing stem with respect to the kinetics

in the aceeptov of cleavage

The mutant precursors used in this study acre al1 mutated in the acceptor-stem. As shown here, and elsewhere, the helical structure of the acceptor-stem is important for the interaction with the enzyme as determined by the K, value (M&lain et al.: 1987; Guerrier-Takada et al., 1989; Kirsebom & Svard. 1992). A simultaneous increase in -K, and E,,, might be the consequence of a change in the ra,te of product-release since it has been suggested that

S. Q. Sviird

1030

and L. A. Kirsebom

CCA is involved in this step of the cleavage reaction (Reich et al., 1987; Guerrier-Takada et al., 1989). Indeed, we have evidence which suggests that product-release is a rate-limiting step in the cleavage reaction of the tRNATy’Su3 wild-type precursor (A. Tallsjii & L. A. Kirsebom, unpublished results). However, we also observed an increase in k,,, whereas K, remained unchanged in t,he cleavage reaction of certain tRNA precursors, in particular in the absence of C5 (Table 1, e.g. pSu3A74 and pSu3-5’SS7). Structural studies suggest that the precursor tRNA is unfolded in the enzymesubstrate (ES) complex (Knap et al., 1990). Therefore, it is possible that the acceptor-stem is partially unfolded at some stage during the cleavage reaction, as a result of the formation of the ES complex. If this is the case, disruption of basepairing in the acceptor-stem at and proximal to the site of cleavage could result in an increase in I&,,. This would be consistent with the finding that tRNA precursors with extended acceptor-stems, resulting from additional base-pairing above the site of cleavage, are cleaved with a reduced rate (A. Tallsjs & L. A. Kirsebom, unpublished results). disruption of base-pairing in the Furthermore, acceptor-stem as a result of ES complex formation has been observed in the cognate tRNAG’“, glutaminyl-tRNA synthetase complex (Rould et al., 1989). Also, studies of the dynamics of tRNA1le by nuclear magnetic resonance spectroscopy suggested that the amino-acyl helix is opened for short periods of time cannot

in soluton

(Reid

& Hare,

1982).

Finally,

we

exclude the possibility (discussed above) that the binding of Mg ‘+ is affected by structural changes near the cleavage site, which consequently results in a difference in the rate of cleavage. In conclusion, base-pairing at and near the site of cleavage of a tRNA precursor is of importance for the

kinetics

of

cleavage,

but

is

not

absolutely

required for cleavage by E. coZi RNase P. In addition, our data suggest that several regions of a tRNA

precursor

FLNase P cleavage

are important

in the location

of the

site.

We thank Dr S. Altman and his colleagues for their generous gifts of pJA2 and C5 protein. We also thank Drs S. Altman, S. Burnett and T. Ruusala for reading and suggested improvements of the manuscript. Miss U. Kagardt is acknowledged for her skillful technical assistance. This work was supported by a grant to L.A.K. from the Swedish Research Council.

References Altman. S., Bothwell, A. L. M. & Stark, B. C. (1974). Processing of E. coli tRNATy’ precursor RNA in vitro. Brookhaven Symp. Biol. 26, 15-25. Altman, S., Baer, M.. Guerrier-Takada, C. & Vioque, A. (1986). Enzymatic cleavage of RNA by RNA. Trends Biochem. Sci. 11, 515-518. Bullock, W. O., Fernandez, J. M. & Short, J. M. (1987). XLl-Blue: a high efficiency plasmid transforming RCA Escherichia coli strain with beta-galactosidase selection. Rio. Tech. 5, 376-378.

Burkard,

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