RNAase P is dependent on RNAase E action in processing monomeric RNA precursors that accumulate in an RNAase E− mutant of Escherichia coli

I. Mol. Biol. (1981)

149, 599-617

RNAase P is Dependent on RNAase E Action in Processing Monomeric RNA Precursors that Accumulate in ‘an RNAase E- Mutant of Escherichiu coli BIMAL

Washington

K. RAY AND DAVID

APIRIOK

Department of Microbiology and Immunology Division of Biology and Biomedical Sciences University School of Medicine, St. Louis, MO 63110, U.S.A. (Receioed

1 December 1980)

A mutant strain of Escherichia coli containing a thermolabile RNAase E, an RNA processing enzyme, accumulates a number of immature RNA molecules at a nonpermissive temperature. Some of these molecules are derived from ribosomal RNA transcription units and contain 5 S ribosomal RNA sequences while others are nonribosomal and contain transfer RNA sequences. In order to assess whether or not RNAase E can produce processing cleavages in such precursor tRNA molecules, we isolated monomeric RNA precursors that accumulate in this mutant strain and studied their maturation in cell-free systems. Our results show that RNAase E can introduce a cleavage near the 3’ end of some precursor tRNA molecules thereby allowing RNAase P to process the precursor molecule at its 5’ end.

1. Introduction Studies on the maturation of transfer RNA in Escherichia coli have shown that precursor tRNA molecules (Altman, 1971; Schedl & Primakoff, 1973) are processed by ribonuclease P (Altman & Smith, 1971; Altman et al., 1974; Carbon et al., 1974: Sakano & Shimura, 1975). Precursor tRNA molecules usually contain extra sequences at both the 5’ and 3’ termini of the tRNA molecules (Altman & Smith, 1971: Bikoff &r Gefter, 1975 ; Schedl et al., 1976). Ribonuclease P is capable of removing extra sequences from the 5’ end of the precursor molecules giving rise to the mature 5’ end of tRNA (Altman & Smith, 1971; Robertson et al., 1972). While processing of tRNA at its 5’ end by RNAase P is well established, and while it is clear from a number of observations that other enzymes must participate in tRNA processing, the identification of other processing endoribonucleases in tRNA processing is poorly documented. By comparing tRNA synthesis in mutants deficient in the RNA processing enzymes RNAase III and RNAase E it was shown that both these enzymes, and particularly the latter, play a role in tRNA processing (Apirion et al., 1980). Recently we demonstrated that a precursor containing two tRNA molecules accumulates in an me strain at the restrictive temperature and that this molecule requires RNAase E, as well as other enzymes, for its processing (Ray & Apirion, 599 0022~3836/81/200599-19

$02.00/O

80 1981 Academic Press Inc. (London) Ltd.

1981). To determine if RNAase E is involved in the final cleavages that, lead to a mature tRN.4, we decided to anal,yze small RN&. including no more than a single t,RNA molecule. which accumulate in t,he rot mutant at, a restrictive temperature. Our studies show that RNAase E can indeed introduce maturation cleavages at, the 3’ ends of some precursor tRNA molecules, and only after the action of RNr\ase E can the precursor be processed by RKAase 1’.

2. Materials

and Methods

(a) Hactprial Esch~rrichiu

co/i strains N3421 (rrw3071)

described by Apirion (1978). RNAase E (Misra & Apirion.

.strair/s

and its isogrnic TT~P+counterpart

Strains carrying an rnr-3071 mutation 1980). DlO is mrtB~ run-20 ((:estrland,

XX22

were

have a thermola~bilr 1966).

(b) Emp2r.u Partially purified RNAase E was prepared in this laboratory (blisra & Apirion. 1979). RSAase P was a gift from Dr Sidney Altman. RNAase T, and RNAasc T, were obtained from Sankyo Company Ltd.. Tokyo. ,Japan. Pancreatic RNAase was obtained from Sigma Chemical Co.. St. Louis, U.S.A.

E. coli cells from strain N3421 (me) were grown at 30°C’ in 25 ml of a Tris/glucose/peptonr medium ((iegenheimer rt al., 1977) and at an -j560 value of about 05 the culture was transferred to 44°C and incubated for 40 min. 32 Pi (Yew _ England Nuclear) was then added to the culture to a final concentration of 0.4 mCi/ml. and the cultures were incubated at 44°C for another 30 min. The labeled cells were harvested hy centrifugation and opened in 4 ml of lysis buffer (20 miv-Tris’ HCl, pH 7.4. 10 mM-Na,EDTA, I’!, SDS?. 3 mM-aurintricarboxylir acid) by heating at 100°C’ for 2 min. The nucleic> acids were then extracted by adding an equal volume of phenol (saturated with IO mm-Tris HCI. pH 7%) and shaking for the nucleic acids, 01 vol. 2 Iv-sodium acetat,? I5 min. To the aqueous layer, containing (pH 5.5) and 2.5 vol. 95% ethanol werr added, and the mixture was stored overnight a,t - 20°C. The precipitated nucleic acids were collected by centrifugation, dried under vacuum and resuspended in 10 mM-Tris. HCI (pH 7.5). 321’-labe1ed RNA was then fractionated on a 17.0 cm x 14.0 cm x 0.3 cm tandem polyacrylamide slab gel containing a 129; separating layer and a top 2 cm of 50/;, stacking by Meyhack rt al. (1973). layer. Details of such polyacrylamide gels wer’fb described Elrctrophoresis was carried out at 4°C for 0.5 h at IO0 V foll owed by 4 h at 250 V. The wet gel was then exposed to a Kodak XR-5 film and the RNA bands. located by autoradiography. were excised and eluted with elution buffer (0.04 M-Tris base. 0.3 M-sodium acetate, 20 mMNa,EDTA, 0.1% SDS. pH 7%). The elutcd RNA was precipitated wit,h ethanol in the presence of carrier RNA. I’recipitated RSA was collected by centrifugation. dried under vacuum and further purified by 2-dimensional polyacrylamidr gel electrophoresis as described by Ikemura & Dahlberg (1973) with some modifications. The first dimension was run in 100/b slab gels containing 5 M-urea (acr~lamide/bisarrylamide, 19 : I : Ply0 (w/v) polyacrylamide trtramethylethylenediamine : @lOjO (w/v) ammonrum persulfate) 17 cm long and 1.5 mm thick; each slot was 4 mm wide. The buffer used contained 80 miv-Tris base, 80 rnx-boric acid and 1 mM-Na,EDTA (pH 8.3). The electrophoresis was carried out at 300 V for 30 min followed by 2.5 h at 500 V. After elrctrophoresis. 0.5 cam strips containing the desired 32Pt

Abbreviations

used:

polyethyleneimine-rellulose:

SDS.

fi,

sodium

prerursor

tlode~yl

,5 S ribosomal

sulfate:

KN.4.

T,,

l
T,:

Plr:I-cellulose.

labeled RSAs wcrc excised from this first dimension gel and placed on a clean glass plat,e and gel (17 cm x 14 cm x 0.15 cm) containing 5 M-urea q-as a 20(+, (M./V) polyacrylamide polymerized around it. Electrophoresis was carried out. using the same buffer as used in t)hv first dimension, for 16 h at 600 1’. The wet gel was exposed to Kodak XR-5 film and spots located by autoradiography were excised and RIVA eluted as described earlier. RSA samples were then precipitated with ethanol in the presence of carrier RNA. The precipitated RNAs ~vere collected by centrifugation and washed with ethanol to remove any remaining salts and thvn dried under vacuum. (d) I II vit,ro prowssing

ruactiotls

and

isolations of thr clcla/mye

products

I’urified RSA molecules prepared as described above were dissolved in st,erile distilled water. For analyt,ical purposes, processing reactions generally contained 2 x IO3 cts/min of 32P-lat)eled RNA precursors, 10 mw-Tris. HU (pH %O), 100 mM-NH,CI. 5 mM-MgCl,, 0.1 rnhv Na,EI>TA. 0.1 mM-dithioerythritol and the following amounts of enzymes: RR’Aase E. 125 pg: RSAase I’: I pg; S30 preparations, 200 pg: in a total volume of 50 ~1. Incubations were carried out, at) 37°C for 5 h. The reactions were terminated by addition of a 022 vol. of a buffer containing 500/, (w/v) sucrose. I2 mM-Na,EDTA. 0.14, (w/v) bromophenol blue, 0.5(‘,, SDS in 50 mM-Tris/40 mM-glycine (pH 83). Such processed materials were then analyzed in a So/l W,, tandem polyacrylamide gel containing 7 >I-urea. After electrophorrsis the gels wcr~ soaked in a mixt’ure of methanol/glycerol/water (25 : 5 : 70. by vol.) to remove the urea and t,hrn dried and autoradiographed. was increased to 5(H) ~1 For preparativcl scale processing. the total volume of incubation along with a IO-fold increase of enzymes and a loo-fold increase in the radioact,ive substrate. The incubations were carried out at 35°C’ for 5 h and reactions wcrc terminated by adding a (k% vol. of the same buffer as described above. The digests were then loaded onto a 50/b/12(),, tandem polyacr?lamide gel containing 7 11.urea (17 cm x 14 cm x (b3 cm) and clectrophoresis was performed tor 12 h at I50 V. The wet gel was exposed to Kodak XR-5 film and the R,;VA bands. which were located by autoradiography, were excised and RNA molecules \\;erv eluted and precipitated as described earlier.

(e) Fittgrrpritttitzy

procrdr~rrs

I’urificd RNA samples were digested nit,h RNAase T, and oligonurleotides t)y the min-fingerprinting trrhniqur of \Tolckaert of /I/. (1976) as described Apirion. 1!)79). (f) Rcdigrstiort

ntcnlysis

of RS.4

oligou

were separated earlier (Ray &

uclcotidrs

The oligonurlcotides obtained after RNAase T, fingerprinting were eluted and digest,ed separately lvith RNAase A (Volckaert & Fiers. 1977) and RNAase T2 (Saneyoshi rt al., 1972). The products were analyzed by d-dimensional chromatography. The details of these analyses have been described earlier (Ray & Apirion. 1979). Quantitation of oligonucleotides was pedhrmed by liquid scintillation counting of various spots. Background counts from Ijlank regions wvre subtracted.

-/

(g) :) End atcalysis

I’urified RNA samples were digested with a mixture of RNAase T, (10 units), RNAase A (2.5 pg) and RNAase T, (2 units) in IO yl of 50 mM-ammonium acetate buffer (pH 5.0) and incubated at 37°C for 4 h and then chromatographed on polyethyleneimine-cellulose plates using 0.75 M-NaH,PO, (pH 3.4) (Celma et al., 1977). Commercially available unlabeled markers such as ppppA and pppp0 were used. 32P-labeled authentic markers were prepared by digesting RNA from an E. coli mutant N3525 which lacks 3 processing ribonucleases (Apirion & (iitelman, 1980) and accumulates a large number of primary transcription products (Plautz & Apirion, 1981).

602

B. K. RAY

AND

I). APIRION

3. Results (a) Accumulation

of R&VA molecules

in the me strain

E. coZi strains with a thermolabile Rh’Aase E (me) accumulate several RNA components at the range of 80 to 400 nucleotides at a non-permissive temperature, while the isogenic ~ne’ strain accumulates only the mature RNAs (10 6, 6 S, 5 S. 45 S and 4 S RNA : Apirion, 1978: Ghora & Apirion. 1978; Ray & Apirion. 1980). A major RNA component which accumulates in r/~e strains at non-permissive temperatures is 9 S rRNA which contains 5 S rRZvTA sequences (Ghora 8: Xpirion. 1978,1979). More recently we showed that multimeric tRNA precursors also accumulate in such a strain at the non-permissive temperature and that their processing to mature tRNAs is dependent on RNAase E (Ray &, Apirion, 1981). Although an me strain does not produce any appreciable amounts of 5 S rRNA at the restrictive temperature (Ghora & Apirion, 1979), several Rh’A components accumulate in the 5 S region of the gel (Ghora & Apirion, 1979; Ray & Apirion. 1980). In order to identify monocistronic precursors of tRNA we decided to characterize the molecules in the 5 S region of the gel which accumulate in the me mutant at the non-permissive temperature. To do so, RNA from strain N3421 (me), labeled at 44°C for 30 minutes, was separated in a 5o/b/I2% polyacrylamide gel as described in Materials and Methods, and the RNA in the 4 S to 5 S region of the gel was eluted and further separated in a two-dimensional polyacrylamide gel. Such a separation revealed a large number of RXA molecules as is demonstrated in Figure l(a). When the cells were labeled for relatively longer times, 60 minutes rather than 30, four major RNA species appeared (Fig. l(b), A to D; species D appears as a doublet). These molecules are not seen in an me+ strain after 30 minutes of labeling at 44°C (Fig. 1(c)), Instead, two major spots appeared, both of which are 5 S rRNA. as determined by T, fingerprinting. Therefore, these four RNA species that accumulate in the me strain at the restrictive temperature after labeling for one hour were selected for further analysis and characterization.

(b) The accumulated

RiVA

molecules are processed by RNAase

E

we tested whether these four RKA In the first stage of the characterization. species can be processed by Rh’Aase E, RNAase P and also by an S30 preparation are shown in from an mp+ me+ strain (DlO). The results of such experiments Figure 2. All four RNA species are cleaved by a partially purified preparation of RNAase E. While the digestion of molecules from species A, B and C by RSAase E gives rise t’o a major product which is only slightly smaller than the substrate, the digestion of species D results in four products: two in the size range of tRNS appear in very low yields, and two smaller than tRNA appear in much larger yields. Species B is digested less efficiently by RNAase E as compared to the other species. It is interesting to note that while all the four molecules are substrates for RNAase E none is a substrate for RKAase P. However, when both enzymes RNAase,E and RNAase P were added simultaneously to the processing reactions (Fig. 2) new products appeared when RKAs A, B and C were used, indicating that these RNAs contain Rh’Aase P sites which become accessible only after the action of RNAase E. The products obtained after combined RNAase E and RNAase P

MONOMERIC

PRECURSORS

,-.lST,

IS

AS

me MI-TAKT

A03

10%

FIG. 1. Two-dimensional polyacrylamide gel electrophoresis of small RNAs produced in an me mutant strain of E. coli and also in its isogenic wild-type strain. The E. coli strains N3421 (me-3071) and N3422 (me+ ) were labeled at 44°C as described in Materials and Methods; the products were fractionated on a P-dimensional 10% to 20% polyacrylamide gel. (a) and (b) Represent the RNA molecules in the me strain after labeling for 30 min and 60 min, respectively: (c) represents the RNA molecules in the me+ strain labeled for 30 min at 44°C. RNAs in spots A, B, C and D in (a) and (b) were used in further studies. Only the RNAs in the 5 S region of the gel in the 1st dimension were transferred to the 2nd dimension. Occasionally phosphate appears at the bottom of the gels (see (b)).

-

1

2

3

4

5

FIG:. 2. Electruphuretic separation of the ire c.i/ru products of digestion of RS.4 species A. H. (’ and D. using various enzyne preparations. The KS..! m&c&s tsolated from X3121 (me) were digested separately with purified HSAase E (lane 2). purified KSAIase P (lane 3). KXAase E + tlSAase P (lane 4) and wild-type gel with E coli X30 (lane 5) at 37-C’ for 5 h lane 1 represents undigested precursor molecules. The digests were seprdtd on a 5”,,/IP,, tandem plyacrylamitle 7 wurea Total fs’. co/i KS,-\ was used as a marker and is shown in the vorner lanes. (The litnes arc arranged in the same order in all the panels.)

Nucleotides

tRNA

5s rRNA

M()SC)MEI
PKE(‘I’KSORS

IS

AS

me MI:‘l’.~N’I’

Ml.5

action seem to be somewhat smaller in size than the products obtained after KNXasr E action alone. Species D, however, does not seem to contain a cleavage site for RX;Aase I-‘. because the addition of R?;Aase P to the assay systetn containing RNAase E does not change the cleavage pattern (Fig. 2). Figure 2 also (830) is capable of reducing the size of shows that an ).w+ rnp+ cell extract molrcules A and C further than the mixture of RNAase E and RNAase P and that t,he final products are only very slightly smaller than the products of the combined action of the two enzymes, The final products derived from molecules A, R and (” are in the size range of tRSAs. When molecule 1) was treated with a preparat.ion of S30. only one of t.he two larger products was retained. while all the others were apparently degraded,

((a) K;\‘.-l .spwies 3 and C contuin tRAYA svqwrwes nud wquirr KIV.4anseE nnd RS=losr P for procrssiny To drtjtwnine lvhether. t)hc RNA species A to 1) do indeed cont’ain transfer RN.4 and to know how RKAase E is involved in t.hpir processing. MY c~tlat*act,c,r,izetl thrw molecules and their various processed products. I’rwursor molecule A was digested with purified RNAase IC. a mixture of E. co/i cell extract and RNXasr E and RKAase I’, and with a wild-type fractionated on polyacrylamide gels. The precursor as well as the different products were then treated with RNAase T, and the oligonucleotides separated lry the minfingerprinting trrhnique. Figure 3 demonstrates such a separation for the precursor and the three different products. Each oligonucleotide was further digested with pancreat.ic RSAase and also with RNAase T, separately and fractionated by twodimrlGona1 chromatograph,v. The information obtained from these analyses is summarized in Table 1. The compositional analysis suggests that molecule .A contains tR,SAy’, t,he sequence of which has been determined by Ishikura rt (rl. (19il). Few oligonucleotides like t8. t9 and t13 in Figure 3(a) appear in a very low molar yield and probably do not belong to molecule A : most likely they come from a contaminat,ing molecule. When the precursor molecule is treated by RNAase E and th product obtained after such cleavage is fingerprinted (Fig. 3(b)). the 5’ end of ttw precursor molecule. oligonucleotide t20, remains intact while spot. t,lS. the 3’ rnd. disappears and two new 3’ ends apprar (3’ and 3” in Fig. 3(b)). They differ ow from the other by a single nucleotide. This suggests that RSAase E cleaves near that 3’ end of t,he precursor. (In this fingerprint t12, Fig. 3(b), migrates differently than in Fig. 3(a) because of differential base modification : the various T, digest’s wire prchpared from different preparations of species A RNA4 which seem to contain diff(~rcwt Itbvrls of base modificat)ions. Spots tl7 and tl8 can sometirrws appear in a low molar yield (compare Pig. 3(b) with Fig. 3(a). ( c ) and (d)) which is very likrl? due to inefficient transfer of these oligonucleotides from the first. to the second dimtwsion in the fingerprinting procedure.) When the product obtained aftcar digt,stion with a mixture of RNAase E and RXAase P was fingerprinted (Fig. 3(c)) thcl l)~.~~~~~rsor-sl)e’ific 5’ end disappeared and a new 5’ end was formed while the 3’ ends were identical to those found after RNAase E action alone. This shows. as t~xlwet~t~d. that RSAase 1’ removes the 5’ end extra sequences from the precursor molrc~ule. \Vhelr t,hr precursor is digest,rd with an S30 preparation. the product has

src~ut’llc’t‘s

606

B. K.

KAY

AND

I). APIRIOS

FIG. 3. Fingerprints of RNAase T,-derived oligonucleotides of species A RNA and also those of its various processed derivatives. These molecules contain transfer RNAyr sequences. (a) Represenm the fingerprint pattern of the precursor molecule, species A RNA. (b). (c ) and (d) Represent the products obtained after digestion of the above precursor with RNAase E, RNAase E + RNAase I?. and wild-type E. coli S30 preparation, respectively. The same oligonucleotide numbers were used in Table 1. Oligonucleotide compositions were determined by redigestion of T,-oligonucleot.ides with pancreatic and T, RNAases (see Table 1). Spot t12 in (a) appears as 2 separate spots due to differential hasp modifications. (The RNA used for T, digestion in the different panels was prepared from different batches of cells.) Due to a similar reason t12 in (b) migrates in a different position. t13 and t,l3’ in (a) have identical oligonucleotide composition. Spots t17 and t18 appear in (b) at a very low molar yield probably because this portion of the RNA molecule somehow became resistant to RNAase T, digest,ion or because of inefficient transfer from the 1st to the 2nd dimensions. Notice that spots t17 and t18 appear in relatively higher concentrations in the other panels.

a fingerprint (Fig. 3(d)) similar to the one obtained after digestion with RNAase E and RNAase P except that a single rather than a double 3’ end is formed. The molar yield of this new 3’ is very low probably due to inefficient transfer to the PEIcellulose plate during the fingerprinting procedure. The bases remaining at; the 3’ end after RNAase E digestion are probably removed by an exonuclease in the 830 preparation (perhaps by RNAase D ; Ghosh & Deutscher, 1978) thus forming the 3’ end of the tRh‘A molecule. The nucleotide sequence of the precursor and the

MONOMERI(’

PRECI~RSORS

IS

AS

rnr

607

MI’TAKT

TABLE 1 Cwnpositiorr

Composition

Oligo 110.

of T, oligowucleotides of species A RIVA and of its txwious processed products

Sptacies A R X1\

c:p ( ‘(: p A\(:p l.Gp (‘(‘(:p 1)I)Gp :L4(:p I’AGp (CT2. 1’)Gp ((‘. I). T)Gp A.4.4Gp (C’.I’,. r*)Gp

Product

I

Product

Product

III

+ +

+ + +

+ + +

((‘. I’. AI’)AAGp (cq2, .S(‘)Gp (cq3. IT,)Gp ((‘2, l:,)AGp ((‘. AAAA(‘)Gp ((‘2. 17,. AAU)Gp ((‘* m3. .4A.-ZU)AAGp ((‘*. I’. pA[T)Gp

Suggested sequence ~for tR\‘AS”t _ 1

GP

+ + + + +

CGp AGp YGp & S4CGp WGp DI)Gp .4AGp

+ + +

T&!Gp ;IAAGp TCmro’I’Gp C’ACCGp & ACCCGp CCCCCGp UCCCAGp Ams2i6AAACCGp

AAIXWXiGp

(~7,

3’ ( trrnrinil(

II

C2, AS,,

PGP &Ll)Gp 2% 1 OH

PGP (pSp. C, V)Gp c*. &xi

PGP CCA,,

RNAaxe ‘I’, derived oligonucleotides were eluted from PEI-plates and analyzed after further digestion with panrreatic RSAase and also with RXAase T2, as described in Materials and Methods. Species A RN.4 is spot A in Fig. I. Products I. II and III represent the processed derivatives obtained after digestion of species A RNA with RNAase E, RNAase E plus RNAase P and wild-type E. coli S30 preparation. respectiveIT (see Materials and Methods). Oligonucleotides were numbered according to their mobilitirs in both dimensions. the same numbers were also used in Figure 3 (a) to (d). +, Indicates thr presmcci of oligonucleotides and - , indicates the absence. due to change or removal by virtue of proc.essiny evcants. t .4c,c*ording to Ishikura et ccl. (1971). : These 3 oligonucleotides t8. t9 and t13 appear in a very low molar yield and therefore probably do not belong to the molecule. Oligonucleotide t13 appears in 2 separate spots t13 and t13’ (see Fig. 3(a)) due to unidentified base modification. $ Oligonu(~lrotide tlf, because of its differential base modification appears in different molar yields and also in different locations in T,-fingerprints (see for instance Fig. 3(a) to (d)). /I L4ppearancr of two 5’ and also 3’ termini in the processed products is probably due to cleavage by contaminating nuclrases, or because the processing enzyme can recognize 2 sites in the precursor rn~~lrc~lr (see Discussion). * An unidentified modified V.

various processing sites described here are shown in Figure 4. Notice that after digesting the precursor with RNAase E and RNAase I’, or after digesting it with S30. a major and a minor 5’ end appear, the major being the correct end oftRNAs” (Table I and Fig. 3). This suggests that cleavage by RNAase P is not always accurate (see Discussion).

RNAase E

FIG. 4. Xuc4eotide sequence and secondary structure of transfer RNA?’ precvxsor molecule. Most probable processing sites for RNAase E and RNAase P are indicated with arrows. which are derived from the analysis of the various processed derivatives as described in Fig. 3 and Table 1. All the modified bases shown in this Figure are not always found in the precursor molecule species A RNA. Base modifications in such cases are post-processing events while others occw prior to processing. The sequenw C-l’ in brackets is tent,ative because from our analysis we do not know the exact arrangement of these I banes.

A similar set of experiments as described above were carried out using precursor RNA molecule B. Figure 5(a) represents the T, oligonucleotide pattern of the precursor molecule. Although this molecule contains few modified bases (Table 2) we were unable to identify a tRh’A sequence which it might contain. As has been mentioned earlier only a low level of cleavage products were produced when this molecule was treated with RNAase E and therefore we could analyze only the composition of the T, oligonucleotides derived from species B before and after digestion with R30. The T, fingerprint of the two products obtained after digestion with RNAase E and a mixture of RNAase E and RNAase P is shown in Figure .5(b) and (c), respectively. Figure 5(d) represents the fingerprint of T, oligonucleotides of a product of this precursor B molecule obtained after digestion with an S30 preparation. Compositional analysis of the oligonucleotides of this molecule and those of its processed product is given in Table 2. This precursor

Flc:. 5. RNAase T,-derived oliponucleotides of species H RSA and of its various processed products. (a) The fingerprint pattern of the precursor molecule. species H RNA. (b). ((a) and (d) Those of’ the prodwts obtained after digest,ion with KNAase E, RNAase IX+ ItNAase P. and wild-type E. coli S~O pwparations. respectively. The same oligonucleotide numbers were also used in Table 2. Oligonucleotidr compositions were determined by redigestion analysis of T-oligonucleot~ides with pancreatic and Tz RSaases (see Table 2). Compostions in (b) and (c) could not be determined because of very IOU radioac*tiw cwunts ohtained after processing (see Fig. 2) and therefore they are not, numbered.

molecule was not processed at its 5’ end since it contains a pppGp (t19. Table 2). I’recursor RX.4 molecule (’ and its various digestion products were treated with RSAasr T, and the oligonucleotide patterns obtained are shown in Figure 6(a) to (d). The compositions of the oligonucleotides are given in Table 3. Spot tI8. which contains several modified bases, sometimes appears in two different positions. This could be due to differential base modification. Spot t5 containing the sequence .\-.A-(‘-(; was not always found. When this precursor is treated with RNAase E a nr\v 3’ end is formed and a few spots like t’4. t9 and t16 disappear while the 5’ end, which starts with pppA, remains intact (Fig. B(b) and Table 3). This suggests that RSAasr E processes this molecule at the 3’ end only. Again like in the case of species A RSA (see Table 1) RKAase E created two 3’ ends differing one from

610

B. K

RAY

ANII)

I). XPIKIOS

TABLE 2 Composition

of T, oligonucleotides of species B RNA of its digestion product

and

Oligo no. t1 t2 t,:? t4 t5 tti t7 t8 t9

Gp (‘Gp AGp I’Gp T)AGp A(‘Gp and (‘AGp AAGp (C-2. V’)Gp

t 10

CC. ~,)GP (I’. AI’)Gp AAAAG JI (C2. r‘, AU)Gp (C,. A4(‘. r))Gp (C2, AC. I:,)Gp ((I. I’,)Gp (C’m3. AC. Y, AAAV)Gp ((“3-4, A(‘. U,. Al’. AAIT)Gp

t11 tl” t13 t14 t15 t1ti t15 tlR

((‘.

'r.

$)GJl

+ + + + + + + + + + + +

[‘SOP (A(‘. (‘)X0,

(‘ompositions of T, ~oligonucleot~ides were determined. as dewribed in Materials and Methods. Species B RSA is the spot B RN.4 in Fig. 1. Oligonuc~leotide numbers are according t)o Fig. 5. + , Indicates the pwsenw of oligonucleotides and - indicates the absence due to change or removal b: virtue of processing events.

another by a single nucleotide. RXAase P then removes sequences from the -5 end (t17 in the precursor) and forms a new 5’ end (Fig. 6(c)). A fingerprint of a product obtained after digestion with an S30 preparation has a similar pattern of oligonucleotides (Fig. 6(d)) to the one obtained from a digestion with RXAase E plus RSAase P (Fig. 6(c)) except that a single base from the 3’ end is removed by such an extract and this could be the effect of an exonuclease in the extract. A comparison of the oligonucleotides of molecule C’ with published sequences suggests which has been determined t)y Ohashi that it contains the sequence for tRYA*“” et al. (1976). Interestingly, the precursor to this molecule also starts with a nucleosidr triphosphate (see Table 3). The fourth and the most abundantly available species, I), was also studied by T, oligonucleotides analysis. The fingerprints are shown in Figure 7. This species is apparently comprised of two similar molecules. each containing at least one RNXase E cleavage site, and no RNAase P cleavage site. This species also contains pppUp and could be made from primary transcripts. The two major products obtained after RNAase E digestion (see Fig. 2) share some common sequences (Fig. 7(b) and (c)). While product 1 (Fig. 7(b)) lacks spots t7, tX. t12, t13 and the 3’

MONOMERIC

PRECVRSORS

IN

AS

me MI:TAST

61 I

Frc:. 6. Fingerprints of RNAase T,-derived oligonucleotide of species C RNA obtained from an me strain of’ E. co/i and of its various processed products. (a) The fingerprint pattern of species C RNA. (b). (c) and (d) Those of the processed products obtained with RNAase E, RNAase Ef RNAase P. and wild-type E. wli S30 preparations, respectively. The same oligonucleotide numbers were also used in Table 3. Oligonucleotide compositions were determined by redigestion analysis of T,-oligonucleotides with pancreatic and T2 RNAases and are given in Table 3. Spot t5 in some of our analyses was missing. Spot tlH and t 18’ in (a) have identical base compositions with different base modifications and therefore

appearas separatrspots. end of the precursor. product 2 (Fig. 7(c)) has all these spots but lacks t4, t10. tll. tl6. t20 and the 3’ end of the precursor (Table 4). The product obtained after digestion of species D RK’A with an S30 preparation. has a T, oligonucleotide patt’ern (Fig. 7(d)) somewhat similar to the one of product 1 (Fig. 7(b)) except that t3. t2l t22 and the 5’ end disappear while a new 5’ end is formed. In the analysis of oligonucleotides of the products of molecule D we could not detect the 3’ ends. This could happen if the 3’ end terminates with a GX,,. By comparing the oligonucleotide composition obtained from molecule D with the published tRx\‘X sequences we could not identify any tRNA molecule that could be processed from this R,KL\ species.

(‘ompositio-11

Oligo no. t1 t:! t3 t4 t.5: tti ti tX tq t IO t11 t12 t13 t14g t15 tlti t1i t1st t19 -I :1 1

Species

(’ RXA

c: p (‘Gp AGp I’Gp A~(‘. Gp ((‘. l-)Gp I)AGp rAGp I~l~Gp ((‘. 8). T)Gp ((‘. 1’)AGp (C‘. V*)AGp (C2. l!)AGp ((‘3. lT,)Gp ((‘. IT,. AC. mGV. AlI)($ ((“2. I.Wl,. 1’*)Gp (pppA1’. C3. I’,)Gp ((‘2. A(‘. IT,. QIT. AA#)Gp CL -zs,, Processed

of T, oligonucleotides of species C R;V&4 and of its various processed products Composition Produc~t I

Produc~t

I1

Prodwt

+ + + + + + +

+ + + -

+ + + -

+ + +

+ +

+ + + +

+ + + +

+ + + +

III

Suggested seyuenw for tRNA*‘“t

+ + + -

(‘ompositions of T, ~gmrrated oligonuc~leotities were determined. as described in Materials and Methods. Species C RSA is the spot C RKA in Fig. 1. Product,s I. II and III represent the processed obtained after digestion of species ( RNA with purified RNAase E. purified derivatives RSAase IX+ KSAase P, and wild-type E. mli S30 preparation. respectively. Oligonucleotide numbers are acwrdinp to Fig. 6. + Indicates the presenw of oli~o~~uc~leotitlrs and - indicates the absence rlw t.o c,hangr or removal by virtue of processing events. t Awording to Ohashi u/ r/l. (1976). $ Oligonwleotide td. i.e. A.A(‘Gp. appears either at a low molar yield or is absent in some of our fingerprint anal\&. Oliponucleotidr t18 appears in 2 separate spots. tlR and t18’. (see Fig. 6(b) and ((I)) due to different‘ial base modification. 5 Oligonu~leotities t!, and t 14 appear in a low molar yield and probably do not belong to the molewle. 11Two 3’ termini in the prwessed product UC probably due to the imprecise action of RNAase E (see legcvxl t.o Table 1 ).

4. Discussion The studies reported here suggest that RXAase I3 can remove extra bases from the 3’ end of at least some tRNX precursor molecules, thereby permitting Rrihase I’ to remove sequences from the 5’ ends of these molecules (Figs 2 to 1 and 6 : Tables 1 and 3). It has been observed that primary transcription products of a transfer RNA gene. in all biological systems studied thus far, are usually longer than the mature tRNA (Altman Rr Smith, 1971: Altman, 1975; Smith. 1976). Primary transcription products of tRNAs contain extra bases at both the 5’ as well as the 3’ends of the mature molecule. In E. coli some precursor molecules contain

(0231

922

c22

(d)

as’

021

FIG:. 7. Fingerprints of K?r’Aasr T,-derived oligonucleotides of species 1) RNA from an VW mutant strain of E. coli and of its various processed derivatives. (a) The fingerprint of species D KSA. (b) and (c) Those of 2 major products obtained after digestion with ItNAase E. (d) The product obt,ained after digestion of species 1) RNA with a wild-type E. coli S30 preparation. The same oligonwleotide numberh were used in Table 1. t23 in (b) is not seen in this fingerprint. since it migrated t,oo far towards the left. It itppwrs in other fingerprints.

more than a single tRSA species. During the process of tRNA biosynthesis such longer precursor molecules are processed by a group of enzymes to form the mat,uw tRNA (Altman. 1975). In the case of monocistronic precursors the extra bases at the 5’ end are removed by RNAase 1’ (Schedl et al.. 1976). So far this is the onl,v cnzymr known to ma~ture the 5’ end of tRSA in ti:. coli, and the studies reported here make it) rather likely that’ in E’. co/i RNAase I’ is the only enzyme which matures t,RSA precursors at their 5’ end. Multicistronic tRS.4 precursors are acted lIpon Ii>. several enzymes including both endonucleasrs and cxonucleasr(s) fA41tman. 197.5). Thrw of the four molecules that we have studied in the present investigation contain nucleoside triphosphatrs at their 5’ ends. However, they might not be primary transcription products since we cannot exclude the possibility that the 3’ ends of these molecules are already processed. Such a possibility exists because many of the tRSA molecules are transcribed in multimrric forms (Altman. 1975). We would like to emphasize that while two of the RNA4 molecules studied here can

614

B. Ii.

RAY

ASD TABLE

Composition

t1 t2 t3 t-i w to t7 t8 t9 t10 t11 t12 t13: t14 t15 tl6 tli tl8t t19t t20 t21 t22 t23 t24

4

of T, oligonucleotides of species D RNA of its various processed products

Oligo IlO.

D. APIRION

Species D RNA Gp CGp AGP CCGp CAGp (C. V)Gp V’AGp AUGp DDGp & UUGp (C, n)AGp (AC, VGp I’AAGp (C2, U)A*Gp (IT, AAU)Gp (IT,, AU)Gp CCCAGp (C. I’. AAU)Gp (C,, AC. U,)Gp (C. AC. I’,-,)Gp (Uz. AU, AAU)AGp (C3. U2, AU)AGp (C,. AC, U,)Gp PPPGP (C. AC, U,m,)X,,

Composition Product I

and

Product II

Product III

+ + + +

+ + + -

+ + +

+ + + + + + + +

+ + + + + + + + +

+ + + + + + + +

+ + + + -

+ + + -

+ pAUGp

Compositions of T,-generated oligonucleotides were determined, as described in Materials and Methods. Species D RNA is spot D RNA in Fig. 1. Product I and II are the 2 processed derivatives obtained after digestion of species D RNA with purified RNAase E. Product III is obtained after digestion of species D with a wild-type E. eoli S30 preparation. +, Indicates the presence of oligonucleotides and - , indicates the absence due to change or removal by virtue of processing events. t These oligonurleotides. t5, t18 and tl9, appear in a low molar yield and might not belong to the major molecule. 1 This oligonucleotide, t13. contains an unidentified modified adenosine moiet,y. § Processed 3’ termini were not detected.

give rise in vitro to tRNA molecules, we have no evidence that these are naturally occurring precursors. Indeed, we anticipate that in wild-type cells many of the RNA processing reactions occur before transcription is over (Ghora & Apirion, 1979), and therefore we would not expect to find these RNA species in appreciable quantities in wild-type cells. One of the interesting aspects of this study is that tRNA precursor molecules with extra nucleotides at their 5’ ends which are potential substrates for RNAase I? accumulated in an me strain in the presence of normal levels of RNAaseP. RNAase P could not act on these molecules due to the extra sequences at their 3’ end. When these nucleotides were removed by RNAase E, only then did they become

MONOMERIC

PKECVRSORS

IN

AN me MITTAR‘T

61.5

substrates for RNAase P (Figs 2 to 6). What is so extraordinary about this situation is that apparently only six extra nucleotides at the 3’ end of ptRPiAS” completely prevent the action of RXAase P at the 5’ end of the molecule (see Figs 3 and 4). Another point of interest is that apparently RNAase P can cleave the ptRNAy’ in two positions. The major cleavage site is at the correct 5’ end and a minor cleavage site is three nucleotides upstream (see Figs 3 and 4 and Table 1). That this is unlikely to be an artifact is indicated by the fact that after action of S30 on pt,RNAS” the same two 5’ ends were found as after the action of the RNAase P preparation (compare Fig. 3(c) and (d)). While it. is possible that the RNAase P preparation contained another enzyme, which cleaved the precursor in the second position. it is rather odd that both enzymes were inactive in the absence of RSAase E (see Fig. 2A). After cleavage with RNAase E two 3’ ends were observed (see Figs 3 and 6 and Tables 1 and 3). This brings up the possibility that the processing enzymes RNAase E and RSAase P are less than perfect in their cleavage reactions. Also in the case of the RNA processing enzyme. RNAase III, it’ is likely that it can int.roduce more than a single cleavage in the same substrate. While (iegrnheimer $ Apirion (1980) inferred that when RNAase III processes p16 tRNiA it creates two cuts in a stem where each of the cuts is in one of the strands and the) are four nucleotides apart. Lund et al. (1980) found that the cuts are only two nucleotides apart. One most obvious question that arises from the present investigation is the following : if RNAase E cleavage at’ the 3’ end of tRXA precursors is mandatory for subsequent action of RNAase P at the 5’ end, why do we not see the accumulation of a large number of precursor tRSAs in RNAase E- mutants as observed in RXAase P negative mutants? This could be explained if RNAase E is not the onI> RNA processing enzyme that participates in the processing of tRNA precursors at their 3’ ends. Another possibility is that in some multimeric tRNA precursors after an independent cleavage by RXAase I’ which forms the 5’ end of the molecule. the 3’ rnd of the tRSA is created by a 3’ exonuclease. We believe that in most tRSA precursors the 3’ ends are processed by an enzyme other than RNL4ase E and t.herefonl most of the tRNA molecules in an me mutant are probably matured (&4pirion Pt al., 1980). A similar phenomenon, i.e. dependence of RKAase P cleavage on another enzyme has been observed earlier by Seidman et al. (1975) when they found accumulation of a proline-serine transfer RNA precursor molecule after infecting with bacteriophage T4 a mutant’ &rain of E. coli isolated by Maisurian & Buyanovskaya (1973). Seidman rt a,l. (1975) observed that while tRNAGly, tRNAG’“, tRNALe” and band E are processed normally, the dimeric precursor, proline-serine t,RNA, is not. The! found tha.t t,he tnutant strain is deficient in a 3’ exonuelease processing activit) which is rrquirrd for removing bases from the 3’ end of the precursor followed b! the addition of a CCA,, through the action of tRN,4 nucleotidyltransferase. Similarly. it was inferred in a previous study that from a precursor that contained 16 S rRN-4. t,RNAy’ and tRNA?k (in this order) RiYAase P could remove onl) tRX*4?;. In order for it to remove tRNA7’ the activity of some other enzyme was required (Gegenheimer & Apirion. 1980). Thus it is clear that the function of

RNAase 1’ can be affected dramatically h?; the various parts of an RNA precursor molecule and that in some cases the activity of one processing enzyme is very much dependent on other processing enzymes. That all the molecules studied here contain at least one cleavage site for RSAasc E is not surprising. since we chose molecules that accumulated after reist.ively long pw-iods in the absence of Rh’ilase E. The studies reported hrre also confirm the previous observation ((ihorn & Apirion. 1979) that strains carrying the r)/~-,707l allele do not mature 5 6 rRNA at, elevated temperatures, since neither 5 S rRSA4 nor I6 were found in the T)IP strain at the elevated temperature (see Fig. I ). molecules ahich appear The f(!ur R,NA species studied hew are very interesting in relatircly large quantities in the E. coli cell. Thesr molecules can be used to study in detail the dependence of RSAase I’ on RXAase IX for its activity in some cases. The fact, tha.t t,he processed pr0duct.s of t,hesr four molecules are wr! wsistant, to the wllular nucleases (they withst,ood .5 h digestion with S30: SW Rtbsults. Fig. 2) suggests that they arc normal RN-4 components of the cell and arr most likely t,o he part of thv stable RNA of exponent~ia~lly growing cells. This is of It is important in this course true for species A and (‘, bvhich givr rise to tKNXs. connection to mention that within thr same Iwcursor molrwles the parts that, arca not destined to brcomc~ mature R,NX are nwre susceptil,le to the cellular nurleases than the part that is destined to lwcomr a mature Ri\;A. This is the case with the 9 S rRNA which contains 5 S rRSA (Misra & Apirion. l!W). I’urthcr analysis of these molecules should help in the undrrstanding of the mode of a&ion of RSAaw E and RNAasc I’ and shed some light on the fbwt,ion of the RSAs found in species R and I). LVe are most grateful to Dr S. Altman for providing us with RXAase I’, and Dr Tapan Misra for a gift of RNAasr E. These studies were supported by Public Health Service grants (:&I-2.5890 and (:M-19821 f’rom t,he National Inst.itutes of Health and C.A-24727 from the ,Yational

(!ancw

Institntcs.

Altman.

S. (1971). .\‘atuw

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