J. Nol.

Biol. (1977) 114, 527-543

Two Ribosome Binding Sites from the Gene 0.3 Messenger RNA of Bacteriophage T7 JOAN ARGETSINGER

STEITZ AND Rrra

A. HIZYA?;

Department of Molecular Blophyaic:.u awl Biochemistry Yale University, NQW Haven, Cot/m. /16’Gl(/, TT.R.d. (Keceived 21 December 1976) Ribosome-protected regions have been isolated and analyzed from the bacteriophage T7 gene 0.3 mRNA labeled in V&JO. Two discrete sites which are nearly equally protected by ribosomes are obtained from what- was previously assumed to be a monocistronic message. Use of appropriate T7 deletion mutant RNAs teas allowed mapping of both ribosomo-recognized regions. Site a is positioned very close to the 5’ terminus of the mRNA and is apparently the initiator region for the major gene 0.3 protein, wllich acts to overcome the host DNA restriction system. Site b is located within several hundred nucleotides of the 3’ end of the RNA and probably initiates synthesis of a small polypeptide of unknown function. Both ribosome binding sites exhibit features common to other initiator regions from Escherichia coli and bacteriophage mRNAs. The proximity of site a to the RNase III cleavage site at the left ond of gene 0.3 may explain why processing by RNaso III is required for efficient translation of the major gene 0.3 protein.

1. Introduction When bacteriophage T7 infects Escherichia coli, the phage DNA is first transcribed by the host polymerase, which produces a transcript corresponding to the leftmost 20% of the T7 genome (Hyman, 1971; Summers rt al., 1973; Studier, 1972; Simon & Studier, 1973). This precursor messenger RNA is then cut at specific sites by a host endonuclease, RNase III, to yield relatively stable, apparently monocistronic messenger species (Dunn & Studier, 1973a,b; Rosenberg et al., 1974). Although the translation of most of these early T7 mRNAs is not significantly affected by the cleavage process, one polypeptide is made much more efficiently if the RNA has been cut, (Dunn & Studier, 1975). This is the gene 0.3 protein, which is encoded by the leftmost early mRNA (Studier, 1972,1973b) and is normally synthesized in very large amounts both in viva (Studier, 1972) and i?~ vitro (Dunn & Studier, 1975 ; CondiO & Steitz, 1975). Synthesis of bacteriophage T3 SAMase. which functions comparably to the T7 gene 0.3 prot,ein (Studier & Movva, 1976), is likewise stimulated by RNase 1 II processing of its mRNA (Hercules et al., 1974). The T7 gene 0.3 protein has a molecular weight of about 9000 (Studier, 1972) and acts to overcome the DNA restrict’ion systems of E. COG B and K (Studier, 1975). The 0.3 mRNA appearing in infected cells is approximately 600 nucleotides long, enough to encode a polypeptide with a molecular weight as large as 22,000 (Simon $ Studier, 1973). Analysis of the RNAs and peptides produced by a number of T7 mutants in which portions of the right end of the U-3 gene are deleted has been 627

62X

.J. A. STEITZ

AND

R. .I.

BRYAS

carried out by Simon and Studier (1973); tl1c.y suggested that most, of thus 0.d RNA is translated but that the 0.3 prot,crin might be processed at it,s N-terminus to produce the 9000 molecular weight polypeptidc observtd in infected cells. In an effort both to underst’and how RNase I11 cleavage affects T7 mRKA translation and to locate the beginning of t,he gene 0.3 cistron on its messenger RNA, we have isolated and analyzed ribosome-protected regions from wild type and several deletion mutant 0.3 mRN14s synthesized in viva. Surprisingly, we find that the 0.3 mRNA contains not one but two ribosome binding sites, which are protect’ed from nuclease digestion with nearly equal efficiency. Thr first site, presumahl,y the initiator region

for the previously

of the mRNA.

charact’erized

gene 0.3 prot’ein,

The second site maps within

lies very

the 3’ one-third

close to the 5’ end

of the (I.,? RNA.

2. Materials and Methods (a) Preparation,

of TY early

RNA

species

Stocks of wild type T7 (Studier, 1969) and deletion mutant phage (Simon & Studier, 1973; kindly supplied by F. W. Studier or W. C. Summers) were grown and purified by banding in CsCl essentially as described by Kramer et al. (1974). D159 was grown on E. coli C instead of the usual T7 host, SY106 (obtained from W. C. Summers). 32P-labeled in wivo-synthesized T7 early RNAs were prepared for use in ribosome binding experiments by the following methods. Either the procedure of Kramer et al. (1974) was used exactly as described, or the cell growth conditions were changed as follows: E. coli SY106 was grown at 30°C in a phosphate-limiting medium containing 0.12 M-Tris.HCl (pH 7.5), 0.08 M-NaCl, 0.02 M-KC& 0.02 M-NH,Cl, 3 mM-Na,SO,, 1 InMMgCl,, 0.2 mM-CaCl,, 0.002 mM-FeCl,, ly’, glucose and I”/0 Casamino acids which had boon depleted in phosphate by treatment, with 0.01 M-Mg2+ and l”i;, cont. NH,OH. At a cell density of about I x IO9 to 2 x log, exponential growth ceases because of phosphate starvation. The cells are then directly u.v.-irradiated, infected and labeled, and the RNA is extracted as described by Kramer et aZ. (1974). Polyacrylamide gel fractionation of T7 early RNAs was achieved using either the 3.75% acrylamide gel system described by Kramer et al. (1974) or gels containing a linear gradient (Studier, 1973b) of 3% to 167; acrylamide (40 :I acrylamide to bisacrylamide). The gel buffer was 0.15 M-Tris.HCl (pH 8.6), 0.1% sodium dodecyl sulfate, 0.002 MEDTA; the electrode buffer contained 0.025 M-Tris, 0.19 M-glycine, 0.1 y. sodium dodecyl sulfate (pH approx. 8.3). Electrophoresis in the gradient gel system was performed on 14 cm x 35 cm x 0.1 cm slabs at about 300 V for 10 to 14 h. After autoradiography of the wet gel, radioactive RNAs were eluted electrophoretically (Kramer et al., 1974) with phenol, using 1 to 2 A,,, units of K 17 RNA as carrier. The RNAs were extracted precipitated with ethanol and, if storage was necessary, kept in distilled water at - 20°C. (b) Isolation

of rihsome-protected

regions

Individual T7 early RNAs were bound to ribosomes in 50 or IOO-~1 initiation reactions containing the following: 100 mM-Tris.HCl (pH 7.5), 50 mM-NH,Cl, 6 mM-Mg acetak, unfraction0.25 mM-GTP, 3 mM-2-mercaptoetllanol, 25 A,,, units of charged formylated and the 32P-labeled T7 ated E. coli tRNA/ml, 100 A,,, units of E. coli ribosomes/ml RNA prepared from a 50-ml culture. In the oxperiments reported here, low-salt washed ribosomes (retaining initiation factors) (Steitz, 1973a) from RV (an F- K12 strain (Condit & Steitz, 1975)) were used. Ribosomes from MREBOO, SY106 (an F- B strain (Condit & Steitz, 1975)) and RV F’ W4680 (Condit & Steitz, 1975) gave comparable results, as did salt-washed ribosomes (Steitz, 1973a) supplemented with crude initiation factors. After incubation of the reaction mixtures for 10 min at 38”C, the initiation complexes were trimmed with 5 pg pancreatic ribonucloase/ml and fractionated on sucrose gradients as previously described (Steitz, 1973a). The fractions uThich comprised the 70 S peak, as determined by direct Cerenkov counting, were pooled and the RNA extracted.

BACTERIOPHAGE

T7 mRNA

RIBOSOME

I3ISI)ING

RITES

529

(c) RLVA sequence analysis TIw well-established procedures described by Barrel1 ( 1971) were used for examination and sequence analysis of ribosome binding sites. Fingerprinting was performed either 111 electrophoresis on cellulose acetate (Oxoid) strips at pH 3.5 and then on DEAE paper in 776 formic acid (Barrell, 1971) or by electrophoresis on Cellogel at pH 3.5 in the first climeJlsion followed by homochromatography on 20 cm x 20 cm polyethyleneimine thin layclrs (Brinkmann, Cel 300 PEl) using homomixture c (Barrell, 1971) in the secolltl tlirnellsion. Prodllcts resulting from U, MNase treatment of primary T, oligonuclootides wer(L at~alyzcd 1)~ digestion n-it11 either alkali or pancreatic ribonucleasc~, as roquiretl : likr!\z,iso tlrtx fractionated products of carbodiimide lnodification plus pancreat,ic ribotlltc:lfvtsc~ \vert’ subsequently digtxstcxd either with alkali or pancreatic ribonuclease afttsl tlebloc~king (Barroll, 1971). In all cases analyses were complicated by unequal labeling of tlrta HSA (Kramer & Steitz, 1973) : quantit,ation of products did not provide usrful inforlrrat iotl. nt~d tlit~refore evidoncc frown several types of analyses \vr?s oftf,n rocl1lirr~d t 0 tlct.c~r~nil~ tlnambiguous secluonc~(s. Partial digestion with spleen phosphodiesterase (Ling, 1972) was used to establish the soquc~rrcc-s of the longer pancreatic oligonucleotides. After clution from the original PEI fingc‘rprint, oligonucleotides w\Tcrc’purified to eliminate carrier RNA by chromatography at rooln tcampc%rature on 20 cm >: 40 cm DElZE thin layers (Brinkmann, Co1 300 DEAE) usirlg I.5 31.pyridinium formato, 7 >I-urea (pH 3.5) to develop the plates. After elution (using no carri(‘r RNA) a defined amount (30 pg) of unlabeled ribosomal RNA was added to each oligotlllclrotido. Digestion was at 37°C in 10 ~1 of spleen phosphodiesterase (a kind gift frown I yielded two tetramers, although in different amounts (see Table 2); most, likely a t:xchnicnal problem is responsible, but) the possibility exists that the sequence at this locus is in fact heterogeneous. Thtl oligonucleotides in the T, and pancreat’ic RXa.se maps were next, overlapped 1)~ a~~al~sls of products generated by partial digestion with T, RNase. During this process. it, became apparent tha,t the oligonucleotidrs comprise t.wo dist’inct, “linkage groups”, corresponding to two discrete ribosomr-protectled regions from the gent> 0.3 mRSA. These are designated site a and s&e h in Figure 3. Note that frayed ends art\ ol)servt>d on both sites, w&h about 30 nucleotides falling wit,hin the regions most hi&l>- prot’ected Kay ribosomes. S&e b is distinct)ivc in that’ it is exceedingly rich in long polypurine tracts. Inspection of the fingerprint,s of Figure 2 reveals that’ oligonucleotides from ribosome binding sites a and b are present in nearly equimolar amounts. This patt,ern was consistently observed in a large number of binding experiment’s, even though there ma>- have been variations in the intactness of the input 0.3 mRSA and ribosomes from different E. coli strains were utilized (see Materials and Methods). Moreover, the fr;tction of the total mRNA radioact,ivity cosedimenting wit,h t,he 70 S ribosomes after n&ease trimming was often nearly lo!;, consistent with efficient ribosomr p otection of two 30-nucleot’ide regions from the 600-nucleot,idr wild type mRYA. T, RNase fingerprints of rihosome-bound regions from the gcnc 0.7, 1.1 and 1.3 mR?;As excised from the same gels (not sholvn) were found not to resemble the pattc~rns for t’hc gene 0.3 ribosome binding &es. Kand 5 and Iland 6 RNAs (which originate from the leftmost’ portion of the Ti penomc (Dunn & Studier, 1973a; Minklry & Pribnow, 1973)) bind much less efficic~nt,ly t ha,n the T7 early mRNAs t’o ribosomes and yield complex patterns when the prot&cld material is fingerprinted. ‘l’ht’sc obscrva’tions are ent’ircly compatible with the \\41-est’ablished fact that these two spccics do not encode specific polypeptide producbs (Dunn 8: Studier, 1973a). (1)) Mapping

of the two ribosonle-bin.d~nll

siks

wiry

deletion

mutant

phcqe

To locat’e rillosome binding sites a and b on the gene 0.3 mRNA we have used appropriatt, deletion mutant T7 phages isolated by Studier (1973a,b). The endpoints of these deletions had previously been mapped relative to the T7 genome by Simon & Studier (1973) : in addition, t,he length of the new RN& (Fig. 4) and corresponding changes in the pattern of polyprptide product#s had been determined. The fractionation of the mutant RN& in tho gel system WC ustltl to prcxparti each of these species is shown in Figure 1. \vc ~wga11 I)!- ttxamining ril~osonlc binding to t ht. mutant) 0.3 mRNAs from HI and (‘42. two cleletions which f&e the 0.3 and 0. i’ mRKAs (SW Pig. 4) and product,

altered but funct’ionsl versions of the major gene 0.3 prot,ein (Studier. 19736,1975; Simon & Studier. 1973). The T, RNase fingerprints of Figure 5(a) and (b) clearly show that these RNAs retain ribosome binding sit’c a, but totally lack all oligonucleotides assigned to ribosome binding site 11.Although it is conceivable that altered RNA secondary/tertiary structure in the deletion RNAs could produce these results (see Steitz, 1973b; Lodish, 1975), the simplest interpretation is that sit’e h is located somewhere in the 3’ terminal l/3 of the wild type 0.3 mRNA (see Fig. 4). Next we analyzed ribosome-protected material from the mut’ant (1.3 mRNA of C66, another deletion mutant which fuses the 0.3 and (I.7 mRNAs. However, in this case the new RNA encodes t’wo product’s, the major gene 0.3 protein and a larger peptide containing the carboxyl end of the 0.7 protein (Simon & St’udier, 1973). The fingerprint here (Fig. 5(c)) reveals that oligonucleotides derived from both (1.3 ribosome binding sites are present in the C66 RNA. Ribosomc binding site b can therefore be assigned t’o that’ region of t’he T7 grnome b&a.ecn the left8 ends of the Hl and C66 deletions (Fig. 4). Finally, to position the beginning of the major gene 0.3 cistron more accurately,

BACTERIOPHAGE

T7

mRNS

RIBOSOME

BINDING

SITES

633

I’!(:. 2. ‘I’, (a) ant1 pancrvtltio (1)) RNssr fingerprints of I,ibosomc-prot,r~ctrtl material from wild 1ylw ‘1’7 gvn~~ 0.3 mRNA. The T, finyerprint, (a) was produrcd hp c~lcctrophoresis from right to left ant1 thvn from toI) to bottom as rlrscrihetl in Materials and Mrt~hods. The G and .4-G spots arc’ uot indrdrd on that portion of t,hv fingerprint illustratd. Thp pancreatic fingerprint (h) was obtairwtl by rlrctlophorrsi~ from right to loft and homochromatogritphy on PEI t,hin layers from bottom t, ) top, again as dexxibetl in Materials and Met,hcds. Sequences of the numbered olipouw!eotitl~+ are rcportcd in Tables 1 ancl 2. B intlicatw thv position of the blw markor dye. Hyphens haw bwn omittwl for clarity.

\vc uwd the short mutant RNA from D159. This deletion again fuses the 0.3 and 0.7 mRNAs, but eliminates almost the entire 0.3 gene (see Fig. 4) and produces no dcbcctable pepbidc (F. W. Studier, personal communication). As shown in Figure 5(d). the D159 RNA yields a ribosome binding sit,e fingerprint containing oligonucleotides assigned to site a but none (as expected) from site b. Thus, the leftmost gem 11.3 initint,or region must lie very close to the 5’ end of t’he wild type mRNA. Moreover. since cvcn the 3’-terminal ribosome binding site T, oligonucleotide 3 (Table 1) is clenrlv present’ in the fingerprint, the entire site a sequence must be contained in the D159 Rn’A. Its position can be more exactly localized as described in the Discussion.

534

J. A. 81’E:I’I’Z

,\NI) TABLE

1-L. ;\.

I 0.3 RNAs (Fig. 5). 8 SCY Materials and Methods

4. Discussion We have identified and analyzed t’wo regions from the gent 0.3 mRNA of bact)eriophagc: T7 which are bound in z&-o by ribosomes under conditions of proiein synthesis initiation. By deletion mapping (Fig. 4) we have located sit,e a very close to the 5’ end of the RNA; overlap with a sequence determined by .J. J. Dunn (personal communication) places the central AUG triplet (Fig. 6) just 35 nucleotides beyond the RNase III cleavage site (see section (b), below). Ribosome binding site b appears between the left-hand endpoints of the Hl and C66 deletions (Fig. 4), within the 3’ third of the wild type gene 0.3 mRNA. (a) Comparison

with other ribosome binding

sites

Gene 0.3 ribosome binding sites a and b are shown in Figure 6 along with a sequence determined by Arrand & Hindley (1973) for a ribosome-protected region from short, (500 nucleotides) RNA transcripts synthesized isn ,uitro by E. coli RNA polymerase using a DNA template they believed to be T7. Surprisingly, neither of the in vi,vo mRNA sequences match the previously analyzed region. The discrepancy is explained by the recmt results of F. W. Studier (personal communication), who has found by restriction endonuclease mapping that the strain used by Arrand & Hindley is apparently T3. In fact, the ribosome-protected sit,c from the in vitro T3 RNA does

536

J.

A.

STETTZ

ASI)

IC.

.\.

Iil data of Simon & Stud& (1973) except, where foxshortened, as indicated by the jagged line, near the left end of gcnc 0.7. Entlpoints for the I>159 deletion have been determined by F. W. Studier (personal communicat,ion). The geno 0.3 mRNA is approx. 600 nucleotides (= 1.5 T7 units) long. Numerical map positions for the endpoints of the early RNA species and of the gene 0.3 deletions are reported by Simon & Studier (1973); thoy are not included here since recent restriction endonuclease mapping of the T7 genome (F. W. Studier, personal communication) suggests that they may require slight adjustment. Arrow* indicate sites of RNase III cleavage of the early RNB precursor (Dunn & Studier, 1973n,b). Ribosomes represent regions of ribosome-protected RNA; in the case of site (I, the region has been located exactly, whereas uncertainty in the location of site b is indicated by drawing t,he riboiomt: with dotted lines. Positioning of the coding regions for the 2 gene 0.5 polypeptidej wai deduced a possibly from Simon & Studier (1973) and from the Discussion (section (b)) ; a wavy line indicates processed segment and dotted lines indicate uncertainty in the exact location of protein tcrmini.

BACTERIOPHAGE

T7 mRNA

RIBOSOME

BINDING

SITES

537

show limited homology with in V&JOT7 site a (16 nucleotides indicated by - - - - - - in Fig. B), despite the fact that the two sequences predict totally different N-terminal amino acid sequences. This suggests that the Arrand & Hindley site corresponds to the beginning of the T3 SAMase cistron; homology of the T7 gene 0.3 protein with the T3 SAMase is supported by the facts that (1) both are encoded by the leftmost gene of the phage early region (Beier & Hausmann, 1973; Studier, 1972), (2) both function to overcome host restriction, and (3) translation of both is markedly stimulated by RNase ITT cleavage of the early mRNA precursor (Dunn & Studier, 1975 ; Hercules at al., 1974). The three ribosome binding sites have been aligned in Figure 6 on the basis of our current understanding of recognition elements in mRNA initiator regions which evidence that during the initiation function in E. co&. There now exists substantial step of protein biosynthesis the 3’ terminus of 16 S ribosomal RNA forms several Watson-Crick base-pairs with an mRNA sequence centered about ten nucleotides 5’ to t’he initiator triplet (Shine & Dalgarno, 1974; Steitz & Jakes, 1975). Indeed, the T3 in vitro site and T7 gene 0.3 site a both exhibit a five base-pair complementarity (underlined in Fig. 6) centered 11 nucleotides 5’ to AUG triplets which appear in about the middle of each of these ribosome-protected regions. This feature of site a and its sequence homology with the T3 in &ro site predict that the major T7 gene

0.3 protein is translated starting with fMet,-Ala, rat’hc,r than thth possil)le fMrt-Ser encoded closer t’o the 3’ end of site a,. The alignment of site b in Figure 6 relative to the other t)wo sequences IYRS :I more difficult problem. Both BUG triplets in site h are preceded by long, appropriately situated polypurine tract’s. For two reasons we suggest that the .4UG closer to the 3’ end of the site h protected region is a more likely initiator codon (see section (b), below). First, in the many previously analyzed ribosome binding sites which contain two or more potential initiator t)riplets, the one utilized is invariably that preceded by the stronger complementarity to 16 S rRNA (see Steitz, 1977). Here (presuming the sequence is correct as writ’ten) pancreatic oligonucleotide 1 ca,n form one more GC base-pair with 16 S rRNA than pancreatic oligonuclcotide 2. Second, although the sequence of T, oligonucleotide 5 has not been established, its mobility on polyethyleneimine cellulose indicates that it is 11 to 13 nucleotides long. This places the rightmost AUG somewhat 3’ to the middle of the entire protected segment, as it routinely appears in ribosome-bound mRNA regions (Steitz, 1977). The assignment is further supported by observed higher yields of pancreatic oligonucleotide 1 relative to pancreatic oligonucleotide 2, which arises from the periphery of the site b prot)ected region. (b) Correspondence between the two ribosome bindirq

sites and gene 0.3 peptides

In previously published work, the wild type T7 gene 0.3 mRNS was identified as the message for one polypeptide product (Studier, 19733), a 9000 molecular weight protein which is required to overcome the host DNA restriction system (Studier,

BACTERIOPHAGE

T7 mRNA

RIBOSOME

BINDING

SITES

539

FIGURE 5(c).

1975). In their study of deletion mutants, Simon & Studier (1973) concluded that this 0.3 protein represents less than half the coding capacity of the 0.3 mRNA and found several deletions (including C66) which enter t’he 3’ end of the mRNA without affecting this protein. Since every protected site so far obtained from native mRNA using E. c& ribosomes has proven to be a true initiator region (St’eitz, 1977), our identification of two ribosome binding sites in the 0.3 mRNA suggests that’ two proteins may be made from this mRNA, the major 0.3 protein st’arting from site a and a second polypeptide starting from site 6 (Fig. 6). This would be consistent with all previous results and also provides a simple explanation for t’he puzzling observation made by Simon & Studier (1973) that infection by the deletion mutant C66 (which fuses the 0.3 and 0.7 mRNAs) produces both the wild type 0.3 protein and a new peptide of molecular weight 35,000. The new peptide would represent the fusion of the 1\;terminus of the site b polypeptide with the C-terminus of t,he (I.7 protein. Further support for the idea that’ a polypeptidc is init’iated ill viz10 at site h is provided by more recent observations made by F. W. Studier (personal communication). Examination of the low molecular weight polypeptides found after infection

FIG. 5. T, fingerprintx of ribos~)mo-~~r~~t~~(~tt~(lregions f&m I hv gvn