translation oftheleader region escherichia tryptophan...

JOURNAL OF BACTERIOLOGY, Mar. 1978, p. 1457-14660021-9193/78/0133-1457$02.00/0Copyright © 1978 American Society for Microbiology

Vol. 133, No. 3

Printed in U.S.A.

Translation of the Leader Region of the Escherichia coliTryptophan Operon

G. F. MIOZZARI AND C. YANOFSKY*Department of Biological Sciences, Stanford University, Stanford, California 94305

Received for publication 17 October 1977

When the trp operon of Escherichia coli contains either of two deletions thatfuse the initial portion of the leader region to the distal segments of the trpEgene, novel fusion polypeptides are produced. The new polypeptides are synthe-sized efficiently both in vivo and in vitro, and their synthesis is subject torepression by trp repressor. Fingerprint analyses of tryptic and chymotrypticdigests of the new polypeptides show that both contain trpE polypeptide se-quences and, despite their different sizes, share the same N-terminal sequence.Our results suggest that synthesis of the new polypeptides is initiated at theAUG-centered ribosome-binding site in the leader region and proceeds in phaseto the region coding for the C-terminal end of the trpE polypeptide.

Polycistronic mRNA transcribed from thetryptophan (tip) operon ofEscherichia coli con-tains a leader sequence of 162 nucleotides pre-ceding the translation initiation codon for thetrpE polypeptide, the first major polypeptidespecified by the operon (3). A short transcriptalso is produced; it contains only the first 142nucleotides of ttp mRNA and results from tran-scription termination at the attenuator, a regu-latory site in the leader region of the operon (1).In vitro studies revealed that RNA transcribedfrom the leader region contains a ribosome-bind-ing site that could serve as a site for initiation oftranslation (14). The nucleotide sequence of theleader region of the tip operon has been deter-mined (21; F. Lee, K. Bertrand, G. Bennett, andC. Yanofsky, J. Mol. Biol., in press); it indicatesthat there is an AUG codon at positions 27 to 29in the center of the ribosome-binding site, fol-lowed in phase by a UGA translation termina-tion codon at positions 69 to 71. This sequencecould code for a leader peptide 14 amino acidresidues in length (Fig. 1). Translation of thisleader segment has been postulated to play animportant role in regulating transcription ter-mination at the attenuator (10).To determine whether leader RNA is trans-

lated, we analyzed the polypeptide products syn-thesized both in vivo and in vitro from segmentsof the tip operon contsining either (i) the entireinitial portion of the operon including the leaderregion, tipE and t7pD, or (ii) the same portionwith deletions that fuse the operator-proximalpart of the DNA sequence coding for the hypo-thetical leader peptide to the distal portion oft7pE. We found that the operons carrying thedeletions directed the synthesis of fusion poly-

peptides, each of which contains the N-terminalend of the hypothetical leader peptide and theC-terminal end of the trpE protein. Schmeissneret al. (18) have also obtained evidence indicatingthat the ribosome-binding site specified by theleader region of the tip operon may be used invivo to initiate polypeptide synthesis.

MATERIALS AND METHODSBacterial strains, phage, and plasmids. All bac-

terial strains used in this study were derivatives of E.coli K-12 W3110. The tip operon internal deletionsAtrpLE1413 and AtrpLE1417 were isolated by Ber-trand et al. as described previously (1). The regions ofthe operon deleted in these strains are indicated inFig. 1.The plaque-forming tUp transducing phage

XcI857trpED1OALE1413 and AcI857trpED10ALE1417were constructed by lysogenizing bacteria containingthese trp operon deletions with XcI857trpED10 (5).After heat induction, the lysates were screened forphage containing trpD but lacking trpE. Up to 50% ofthe progeny were found to have acquired the deletions.

Plasmid pVH153 was kindly provided by V. Hersh-field and D. Helinski. It contains a 4.7-megadaltonEcoRI fragment carrying tpPOLED"C" from thephage XtrpED10 inserted into the EcoRI site in pVH51(mini-CoEl; 7). Plasmids pGM1 and pGM3 were con-structed in the course of this study. They are identicalto pVH153 except that they carry the deletionsAtrpLE1413 (pGM1) and AtrpLE1417 (pGM3). A dia-gram of plasmids pVH153, pGMl, and pGM3 is shownin Fig. 2. Plasmid DNA was prepared as described inSelker et al. (19). The preparation of phage and phageDNA has been described (25).

Buffers and solutions. The makeup of the buff-ers and solutions used was as follows. The polyacryl-amide gel electrophoresis sample buffer contained 60mM tris(hydroxymethyl)aminomethane-hydrochlo-ride buffer (pH 6.8), 3% (wt/vol) sodium dodecyl sul-

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1458 MIOZZARI AND YANOFSKY

Met- Lys-Ala -lie - Phe-Val - Leu-Lys -Gly-Trp-Trp-Arg-Thr-Ser

I F N

trp PO trp L trp E trp DfIleader attenuatorpeptide

ErIF1417trp L LE 141 I

trp_A LE 1413

FIG. 1. Initial portion of the trp operon of E. coli. The bottom part of the figure is a simplified map of thetrpPOLE region showing the approximate positions of the attenuator and the sequences coding for thehypothetical leader peptide. trpL and trpE are not drawn to scale. The nucleotide sequence of the RNAtranscribed from the initialportion of the leader region is shown at the top of the figure. It shows the locationof the ribosome-binding site (14) and the amino acid sequence of the hypothetical leader peptide. The blackbars at the bottom of the figure indicate the extent of the internal deletions AtrpLE1417 and AtrpLE1413. Theleft-hand end point of each deletion has been determined to fall between base pairs 49 and 52 of the leaderregion; the right-hand deletion end points were located by genetic recombination analyses with known trpEpoint mutants (1; Bertrand and Yanofsky, unpublished data).

Eco RII~

Eco R' / /

HPaIp

FIG. 2. MAP ofplasmid pVH153 showing the lo-cation of trpPOLED"C" and the sites of cleavage byrestriction endonucleases EcoRI and Hpa I. TheplasmidspGM1 andpGM3 are identical to pVH153,except that they carry the trp operon internal dele-tions AtrpLE1413 (pGM1) and AtrpLE1417 (pGM3).The zigzag line next to trp"C" marks the boundarybetween bacterial trp operon sequences and phageDNA.

fate (SDS), 5% (vol/vol) 2-mercaptoethanol, 15%(vol/vol) glycerol, and 0.05% (wt/vol) xylene cyanolFF. The staining solution contained 50% (vol/vol)methanol, 10% (vol/vol) acetic acid, and 0.25%(wt/vol) Coomassie brilliant blue. The destaining so-lution contained 25% (vol/vol) ethanol and 10%(vol/vol) acetic acid. The elution buffer X contained0.5 M NHI acetate, 0.01 M Mg acetate2, 0.1 mM

ethylenediaminaetetraacetate, and 0.1% (wt/vol)SDS.

In vitro protein synthesis. The preparation ofS30 extracts and the composition of the reaction mix-ture for in vitro protein synthesis were essentiallyas described in ZaJkin et al. (25), except that thereactions were conducted in the presence of polyethyl-ene glycol (Carbowax-6000, Union Carbide) at a finalconcentration of 25 mg/ml. For labeling experimentswith [3S]methionine and [3H]lysine, the correspond-ing unlabeled amino acids were either omitted fromthe reaction mixture (methionine) or reduced to 10%of the normal concentration (lysine). After 60 min ofincubation at 340C, incorporation was terminated bythe addition of the corresponding unlabeled aminoacid to a final concentration of 5 mM; ribonuclease(RNase) A (50 itg/ml) and deoxyribonuclease (DNase;50,ug/ml) were then added. Incubations were contin-ued for 5 min at 34°C before the reactions were chilledon ice. Protein was precipitated by the addition of 9volumes of cold acetone and pelleted by centrifuga-tion. The pellet was dissolved in polyacrylamide gelelectrophoresis sample buffer by heating at 950C for2to3mi.Phage infection of UV-irradiated cells. UV-ir-

radiated cells (13,000 ergs/mm2) of the UV-sensitiveE. coli K-12 strain 159 (12), carrying either the trpR+or the trpR allele, were lysogenized with AcI587(ind)and infected with purified Atrp transducing phages inthe presence of tryptophan (50 yg/ml), as describedby Ray and Murialdo (16). Proteins synthesized be-tween 15 and 30 min after infection were labeled with[3S]methionine (10 liCi/ml; 315 Ci/mmol). Incorpo-ration of the labeled amino acid was stopped by theaddition of unlabeled methionine to a final concentra-tion of 5 mM, followed by sodium azide (50 mM). Thecultures were chilled on ice and immediately pelleted

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LEADER REGION TRANSLATION 1459

by centrifugation. The pellet was lysed directly inpolyacrylamide gel electrophoresis sample buffer byheating for 3 min at 950C.

Polyacrylamide gel electrophoresis. Proteinslabeled in vivo or in vitro were generally separated on12.5% polyacrylamide gels containing 0.1% SDS by thediscontinuous buffer method described by Laemmli(9). In some instances (see figure legends), 10 to 20%linear polyacrylamide gradients containing 0.1% SDSand 8 M urea in both the separating gel and thestacking gel were used. After electrophoresis, the ra-dioactive bands were located directly by autoradiog-raphy for preparative runs (see fingerprint analysisbelow). In analytical runs, the protein bands werefixed by immersion in staining solution for 2 h and thedestained for at least 4 h with several changes ofdestaining solution. The destained gel was brieflyrinsed in 25% ethanol to remove excess acetic acid anddried on filter paper. The radioactive bands werelocated by autoradiography ([3S]methionine) or fluo-rography ([3H]lysine) (2).The following polypeptides were used as markers

for the determination of the molecular weights oflabeled proteins (molecular weights in parentheses):horse heart cytochrome c (12,400), lysozyme (14,300),sperm whale myoglobin (17,800), immunoglobulin L-chain (23,500), tryptophan synthetase a (28,700), oval-bumin (45,000), immunoglobulin H-chain (50,000), andbovine serum albumin (67,000). With the exception oflysozyme (Worthington Biochemicals Corp.) and tryp-tophan synthetase a (prepared in this laboratory), allmarkers were from a set purchased from Mann Re-search Laboratories. The molecular weights of immu-noglobulin H- and L-chains are taken from Weber andOsborn (23).

Fingerprint analysis of tryptic and chymo-tryptic digests. Radioactive bands were located ongels by autoradiography. Gel slices containing thelabeled proteins were fragmented into small pieceswith a glass rod. Portions (1 to 2 ml) of elution bufferX and bovine serum albumin (50,g) were added, andthe mixture was incubated at 370C overnight. Thebuffer solution containing most of the labeled proteinwas separated from the gel pieces by filtration througha pipette tip plugged with glass wool. The proteinswere precipitated four times by adding trichloroaceticacid to 20%, suspending in 0.1% NH40H, and washingthree times with acetone. The dried residue was takenup in 50,l of performic acid and oxidized for 60 min at0°C. The acid was removed by several cycles of wateraddition and drying in a desiccator. Trypsin and chy-motrypsin digestions were performed in 0.05 MNH4HCO3 (pH 7.8) for 4 to 10 h at 370C. The volatilesolvent was removed by drying in a desiccator, and thedry residue was taken up in a small volume of water.The amount of radioactivity in the final sample wasdetermined by counting a small sample of the solutionin a liquid scintillation counter.

Material corresponding to approximately 200,000cpm was applied to Whatman 3MM paper and finger-printed as described by Helinski and Yanofsky (6),except that electrophoresis was used in the first di-mension. The labeled peptides were located on thefingerprints by autoradiography.Enzymes and chemicals. Alpha chymotrypsin

and trypsin-TPCK were obtained from WorthingtonBiochemicals Corp. (Freehold, N.J.). Restriction en-donuclease EcoRI was purchased from Miles ResearchLaboratories (Elkhart, Ind.). Restriction endonucleaseHpa I and T4 DNA ligase were supplied by NewEngland Biolabs (Beverly, Mass.).

L-[35S]methionine (315 Ci/mmol) and L-[4,5-3H-(N)]lysine (39 Ci/mmol) were obtained from NewEngland Nuclear Corp. (Boston, Mass.). SDS waspurchased from Bio-Rad Laboratories (Richmond,Calif.) and recrystallized twice from ethanol before usein gel electrophoresis.

RESULTSConstruction of phage and plasmids

carrying trpLE deletions. Bertrand et al. (1)have reported the isolation of mutants with trpoperon internal deletions that have one endpoint in the leader region of the operon and theother in one of the five structural genes. Amongthe mutants isolated were two strains, W3110AtrpLE1417 (1) and W3110 AtrpLE1413 (K. Ber-trand and C. Yanofsky, unpublished data), thathad their left-hand deletion end points (Fig. 1)within the region coding for the hypotheticalleader peptide and their right-hand deletion endpoints within the first major structural gene,trpE. The left-hand end points of both deletionswere determined to be between base pairs 49and 52 of the leader region by oligonucleotideanalysis of 32P-labeled operator-proximal trpmRNA (1; Bertrand and Yanofsky, unpublisheddata). The deletion strains retain the AUG-cen-tered ribosome-binding sequence early in theleader region as well as the initial seven or eightcodons for the hyopthetical leader peptide (Fig.1). In the absence of functional trp repressor,i.e., when a trpR allele is introduced, both strainsshow an 8- to 10-fold increase in the level oftrpB protein compared with the level in an oth-erwise isogenic trpR trp+ strain (1; Bertrand andYanofsky, unpublished data). This increase indistal gene expression results from the removalof the attenuator, a site of transcription termi-nation in the leader region of the operon (1).The levels of trp operon expression observed inW3110 trpR strains with AtrpLE1413 orAtrpLE1417 are among the highest observed inattenuator deletion strains, indicating that thetwo deletions do not have polar effects on distalgene expression. Because the two deletions ter-minate in regions of trpE within which nonsensemutations cause severe polarity (24), it seemslikely that they allow in-phase fusion of trans-lated segments of leader tip mRNA and trpEmRNA. Alternatively, the deletions could gen-erate efficient translation restart sites withintrpE. In either case, the carboxy-terminal seg-ment of the tipE polypeptide should be synthe-sized.

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To analyze the polypeptide products synthe-sized from operon segments containingAtrpLE1413 and AtrpLE1417, the deletions weretransferred onto phage AcI857trpEDlO by ge-netic recombination. Subsequently, a restrictionendonuclease EcoRI-generated fragment of thisphage DNA containing the operator-proximalpart of the tip operon was cloned, with pVH51(mini-ColEl; 7) as a vector. Both resulting plas-mids carry trpD+ and, therefore, can be selectedfor on the basis of their ability to allow strainW3110 AtrpLD102 to grow on anthranilate (8).Figure 2 shows a diagram of pVH153, a plasmidthat carries the initial segment of the wild-typetrp operon up to the beginning of trpC (5), anda diagram of the isogenic plasmids, pGM1 andpGM3, that carry the deletions AtrpLE1413(pGM1) and AtrpLE1417 (pGM3). All of theplasmids have two recognition sites for restric-tion endonuclease Hpa I; one is in the trp pro-moter/operator region, and the other is justbeyond the trp genes in phage DNA (unpub-lished data from this laboratory). The Hpa Idigestion pattern of the three plasmids is shownin Fig. 3. Although the larger fragment (molec-ular weight, 4.3 x 106) is the same in all cases,the smaller fragment, which has a molecularweight of 2.5 x 106 in pVH153, is reduced in sizein plasmids pGM1 (molecular weight, 1.75 x 106)and pGM3 (molecular weight, 2.05 x 106), re-flecting the sizes of the respective deletions. Thedifferences in molecular weight indicate that thedeletions remove about 1,200 base pairs(AtrpLE1413) and 720 base pairs (AtrpLE1417).Expression of plasmid DNA in vitro.

Faithful expression of the tip operon DNA of Xor )80 trp transducing phages in a coupled invitro transcription/translation system (S30) hasbeen reported previously (25). Depending on thenature of the template used, it was found thattranscription of the trp genes could originate ateither a phage promoter or at the tip promoter.However, only trp-promoted transcription wassensitive to the presence of trp repressor. Plas-mids pVH153, pGM1, and pGM3 were used astemplates in the in vitro transcription/translation system. The proteins made in thepresence or in the absence of trp aporepressorwere labeled with [35S]methionine and separatedon a SDS-polyacrylamide gel. The results (Fig.4) suggest that transcription of the trp genes inplasmid pVH153 initiates exclusively at the trppromoter, as synthesis of the trpE and trpDproteins is essentially eliminated if the S30 ex-tract is derived from a strain containing func-tional trp aporepressor (trpR+). Figure 5 showsan autoradiogram of a dried gel. Whereas boththe trpE (molecular weight, 60,000) and trpD(molecular weight, 65,000) polypeptides are

FIG. 3. Agarose gel electrophoresis of plasmidDNA restricted with restriction endonuclease Hpa I.Restriction digests ofplasmids pGM3 (1), pGMJ (2),and pVH153 (3) were run on 0.8% agarose gels inTEA-NaCl buffer as described previously (19). Theoutside slots (V contain A DNA restricted with restric-tion endonuclease EcoRI; the fragments serve asmolecular weight markers (22).

present and strongly repressed when pVH153DNA is provided, there is no band in the positionof the trpE polypeptide with the deletion plas-mids as templates. In each case a new, veryintense band appears that is strongly repressedby the presence of tip repressor. The new poly-peptide bands migrate with molecular weightsof 19,000 (pGM1) and 36,000 (pGM3). The dif-ference in molecular weight between the tipEprotein and the new polypeptides generated bythe AtrpLE1413 and AtrpLE1417 deletions cor-relates well with the amount of DNA deleted inthese strains (see Discussion). The new proteinbands could thus represent in-phase fusions be-tween the N-terminal end of the putative leaderpeptide and the C-terminal end of the trpEpolypeptide. We will refer to these putative fu-

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polypeptide band (molecular weight, 65,000) andone additional very intense polypeptide band.The additional bands have mobilities corre-

sponding to molecular weights of 19,000 for theprotein specified by AtrpED1OALE1413 and36,000 for the protein of AtrpED1OALE1417;they are identical in size to the presumed trpLEfusion proteins observed in vitro (Fig. 5). Al-though only trp-specific products are synthe-sized after infection of a A lysogen withAtrpEDALE1417, A-specific protein synthesis isonly partially repressed when the phage em-

ployed is AtrpED1OALE1413. This incompleteregression may be related to the fact thatAtrpED1OALE1413 makes clear plaques on sen-

sitive bacteria at 32°C and, thus, is not normallyrepressible.Analysis of trypsin and chymotrypsin di-

gests of trpLE fusion proteins. Polypeptidessynthesized in vitro with plasmids pVH153,

Increasing molecular weight

FIG. 4. Repression of the synthesis of trp enzymes

in vitro. Proteins synthesized with pVH153 as tem-plate were labeled with [JS]methionine (150 ,uCi/ml,315 Ci/mmol) in vitro with S30 extracts derived fromstrains carrying either the trpR' or trpR allele. Thelabeled proteins were separated on a 10 to 20% gra-dient polyacrylamide gel containing SDS and 8 Murea and were located by autoradiography of thedried gel. The figure is a densitometer scan (Joyce-Loebl) of the autoradiogram; it shows the labeledproteins synthesized in the presence (trpR+) or ab-sence (trpR) offunctional trp repressor.

sion proteins as LE1413 and LE1417 polypep-tides.In vivo expression of trp operon seg-

ments containing trpLE deletions. UV-irra-diated E. coli cells were infected with AcI857 or

Atrp transducing phages carrying deletionsAtrpLE1413 or AtrpLE1417. Proteins synthe-sized after phage infection were labeled with[3S]methionine and analyzed by electrophoresison SDS-polyacrylamide gels. UV irradiation es-

sentially eliminates host protein synthesis (slot1) without serious damage to the RNA or proteinsynthesis machinery (slot 3) (Fig. 6). If the in-fected host is a AcI857(ind) lysogen, the expres-sion of the A genes of the infecting phage isprevented (slot 4). In addition, transcriptionfrom the trp promoter can be repressed by in-

fecting a host carrying a functional trp repressorin the presence of tryptophan (slots 5 and 7).

Infection of trpR+ and trpR A lysogens withAtrpED1OALE1413 and AtrpEDJOALE1417identifies two protein bands that are present inthe trpR but not in a trpR+ host: the trpD

trp D4-trpE

LE1417

mOR LE 1413

la lb 2a 2b 3a 3b

R _ R+ R R _

FIG. 5. Polyacrylamide gel electrophoresis ofpro-teins labeled in vitro. Proteins synthesized in vitro

with pVHIJ53 (la, lb), pGMI (2a, 2b), andpGM3 (3a,

3b) as templates were labeled with /'S/methionine(250 jiCi/ml, 315 Ci/mmol). The S30 extracts were

derived from either trpR' (la, 2a, 3a) or trpR strains

(lb, 2b, 3b). The labeled proteins were separated on

a 12.5% polyacrylamide gel and located by autora-

diography. The arrows indicate the positions of the

trp-specific polypeptides synthesized by the three

plasmids.

trp R

Ii

trp R

NI It rp EtrpD I

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trpE protein. In addition, digestion with eithertrp D enzyme yields a prominent spot that is identical

for both the LE1413 and LE1417 polypeptidesbut that is not present in the digest of the trpEprotein (indicated by an arrow in Fig. 7). Be-cause the C-terminal end of both fusion proteinsmust be identical to the C-terminal end of the

LE1417 trpE polypeptide, the additional common spot~< " must arise from the N-terminal portion of the

putative fusion proteins. This suggests that theLE1413 and LE1417 polypeptides share thesame N-terminal sequence.

In a similar experiment in which [3H]lysinewas used to label proteins, the same N-terminaltryptic and chymotryptic peptides observed with

LE1413 [35S]methionine as label appeared on finger-Ut prints of the LE1413 and LE1417 polypeptides

but not on fingerprints of the trpE polypeptide(results not shown), indicating that the N-ter-minal tryptic and chymotryptic peptides of theLE1413 and LE1417 fusion proteins containboth methionine and lysine.

1 2 3 4 5 6FIG. 6. Polyacrylamide gel electrophoresis

(12.5%) of the proteins labeled 15 to 30 min afterinfection of UV-irradiated cells with Atrp transduc-ing phage. All samples with the exception of no. 2(unirradiated control) were irradiated with UV at13,000 ergs/mm2. The slots show: (1) uninfected con-

trol, (2) unirradiated control, (3) strain 159 infectedwith AcI587, (4) strain 159 (AcI857) infected withAcI857, (5) strain 159(AcI857) trpR+ infected withAcI857trpED10ALE1413, (6) strain 159 (AcI857) trpRinfected with AcI857trpED10ALE1413, (7) strain 159(;c1857) trpR+ infected with XcI857trpED1OALE1417,and (8) strain 159 (Ac1857) trpR infected withAcI857trpED10ALE1417. The arrows indicate the po-sitions of the trp-specific polypeptides synthesized bythe two trp transducing phages.

pGMl, and pGM3 were labeled with [3S]methi-onine and separated by electrophoresis on SDS-polyacrylamide gels. The bands correspondingto the trpE protein and to the putative fusionproteins LE1413 and LE1417 were located byautoradiography, cut out, and eluted from thegel. After oxidation with performic acid, theproteins were digested with trypsin or chymo-trypsin, and the digestion products were ana-lyzed by fingerprinting with high-voltage elec-trophoresis in the first dimension and descend-ing chromatography in the second dimension.The results of this analysis are shown in Fig. 7.It is evident that the majority of the [3S]methi-onine-labeled peptides generated by trypsin orchymotrypsin digestion of the fusion proteinsare also present in the respective digests of thetrpE protein. Therefore, the LE1413 andLE1417 polypeptides must contain part of the

DISCUSSIONTwo internal deletions in the tip operon of E.

coli, AtrpLE1413 and AtrpLE1417, have one ter-minus in the leader region of the operon and theother within trpE. Strains carrying either dele-tion retain the DNA segment corresponding tothe AUG-centered ribosome-binding site at thebeginning of the leader mRNA (14), as well as

the first seven to eight amino acid codons for theputative leader peptide. The trp operon DNAcontaining the two deletions was shown both invitro and in vivo to promote the synthesis ofnew polypeptides, each of which is subject torepression by trp repressor and is not coded forby the wild-type trp operon. Both polypeptidescontain trpE sequences and could arise as a

consequence of either an in-phase fusion of theresidual part of tipE to an external translationinitiation site or the activation of efficient trans-lation restart sites within trpE.

Several observations indicate that the new

polypeptides represent the fusion of the N-ter-minal end of the leader peptide and the C-ter-minal end of the trpE protein.

(i) The molecular weights of the postulatedLE fusion proteins are consistent with theamount of DNA removed by each of the dele-tions. A restriction analysis of isogenic plasmidscarrying either wild-type trpE or the trpLE dele-tions showed that AtrpLE1413 and AtrpLE1417remove about 1,200 and 720 base pairs, respec-tively. Thus, these deletions reduce the codingcapacity by 400 and 240 amino acids, and one

would expect the molecular weight of the trpEpolypeptide to be reduced from 60,000 to about18,000 for the AtrpLE1413 deletion and to about

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35,000 for the AtrpLE1417 deletion, if the re-maining trpE sequences are translated. The cal-culated values of 18,000 and 35,000 are virtuallyidentical to the observed molecular weights(19,000 and 36,000) ofthe new polypeptides spec-ified by the two deletions, both in vivo and invitro. This suggests that the residual segment oftrpE in each of the deletions must be translatedstarting at the site immediately preceding orfollowing the fusion point between trpE and theleader region.

(ii) The presumed LE fusion polypeptides aresynthesized at a very high rate. Although bothAtrpLE1413 and AtrpLE1417 have their right-hand deletion end point in regions of trpE withinwhich nonsense mutations have been shown tocause severe polarity (24), neither of the dele-tions has a polar effect on distal gene expressionin vivo. The high levels of trpB protein observedin trpR strains carrying the two deletions suggestthat distal trpE mRNA must be translated effi-ciently in both deletion operons. This conclusionis confirmed by the observation that, under con-ditions ofDNA excess in vitro, between 6 and 10times more [3S]methionine is incorporated intothe fusion polypeptides than into the trpE pro-tein (data not shown). This increase closely re-flects the observed 8- to 10-fold increase in distalmRNA production associated with removal ofthe attenuator site (1) and indicates that initia-tion of translation is just as efficient for the newpolypeptides as it is for the wild-type trpE pro-tein. Reinitiation of translation at intemal siteswithin structural genes after chain terminationhas been described for the rIIB cistron of phageT4 (17) as well as for the Z gene (11, 13) and theI gene (4, 15) of the lac operon of E. coli.However, the efficiency of reinitiation is gener-ally low. The restart sites at the beginning of thelacI gene lead to the accumulation of C-terminalrepressor fragments at 10% of the wild-type re-pressor level (4), and the polar effects of chaintermination mutants in lacZ on transacetylaseexpression (11, 13, 26) suggest similar or onlyslightly higher efficiencies for the restart siteswithin lacZ. If the high levels of trpLE polypep-tides observed with both LE deletions were dueto reinitiation of translation within t7pE, inter-nal reinitiations would have to occur essentiallyat the frequency observed for wild-type trpEmRNA. The existence of two such sites withintrpE is very unlikely, particularly in view of thefact that there appear to be no major discontin-uities in the trpE polarity gradient (24).

(iii) Additional evidence against internal re-starts within trpE is provided by the fact thatboth new polypeptides share an identical se-quence at their N-terminal end. Fingerprint

analysis of tryptic and chymotryptic digests ofboth LE fusion proteins yields a prominent spotthat is identical for both fusions, but is notpresent in digests of the trpE protein. As arguedearlier, these spots must arise from the N-ter-minal portion of the fusion polypeptides. Themost likely way both polypeptides can share thesame N-terminal tryptic and chymotryptic pep-tides is for them to be initiated at the same siteoutside trpE and to have the same amino acidsequence up to the first site of trypsin and chy-motrypsin cleavage. Among the approximately50 transcribed nucleotides that precede the fu-sion point between trpL and trpE (Fig. 1), theonly obvious candidate for this initiation site isthe AUG-centered ribosome-binding site earlyin the leader region. In in vitro experiments, thissite has been shown to bind ribosomes with thesame efficiency as the trpE ribosome-bindingsite (14). As pointed out previously (14), it re-sembles a number of other ribosome-bindingsites in E. coli in that it contains sequences thatcan potentially base pair with sequences at the3' terminus of 16S rRNA (20). Translation ini-tiation at the leader ribosome-binding site wouldfuse the first seven or eight amino acids of theputative leader peptide in phase to the C-ter-minal portion of the trpE polypeptide. In fact,both the tryptic and chymotryptic N-terminalpeptides of the fusion proteins contain both me-thionine and lysine. Because the N-terminal se-quence of the putative leader peptide is Met-Lys-Ala-Ile-Phe-Leu, one would expect both thetryptic peptide (Met-Lys) and the chymotrypticpeptide (Met-Lys-Ala-Ile-Phe) to be labeled byeither [3S]methionine or [3H]lysine if synthesisof the LE1413 and LE1417 polypeptides is ini-tiated at the leader ribosome-binding site.The high rate of synthesis of both new poly-

peptides, the fact that they share a common N-terminal tryptic and chymotryptic peptide(which is not present in digests of the intacttrpE protein), and the existence of an efficientribosome-binding site immediately precedingthe fusion point between trpL and trpE lead usto conclude that the LE1413 and LE1417 poly-peptides represent in-phase fusions of the N-terminal end of the leader peptide and the C-terminal end of the trpE protein. Schmeissneret al. (18) have reported a similar observation ina fusion of the lacI gene to the early part of thetip operon. They sequenced the N-terminal seg-ment of the resulting fusion protein and foundthe first six amino acids to be identical in se-quence to the putative leader peptide of the trpoperon. Our own observations confirm their re-sults and indicate that the ribosome-binding siteat the beginning of the trp leader sequence is

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FIG. 7. Two-dimensionalpattern oftryptic (a) and chymotryptic (b) digests ofthe trpE polypeptide and theLE1413 and LE1417fusionproteins. Electrophoresis (left to right) was used in the first dimension; descendingchromatography (bottom to top) was used in the second dimension. The arrows indicate the positions of thespots that are present in the digests of both fusion proteins but not in the digests of the trpE protein.

used for the efficient initiation of translationunder conditions where the leader peptide isfused to the C-terminal portion of another poly-peptide.

AUl-our efforts to demonstrate the synthesis ofthe intact leader polypeptide itself have beenunsuccessful to date (G. Miozzari, unpublished

data). In particular, samples labeled both in vivoand in vitro for various amounts of time (2 to 60min) were analyzed on polyacrylamide gels (10to 25% with increasing cross-linker concentra-tion, containing 8 M urea to reduce the poresize), on two-dimensional fingerprints (as in Fig.7), and on peptide columns (Dowex 50). In other

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experiments, we tried unsuccessfully to demon-strate the synthesis of the N-terminal portion ofthe leader peptide by analyzing tryptic and chy-motryptic digests of in vitro reactions on finger-prints (as in Fig. 7). We have demonstrated thattranslation of the leader region can be initiatedwith high efficiency, and, therefore, one wouldexpect the leader peptide to accumulate in sig-nificant amounts. Our inability to detect its syn-thesis may reflect very rapid degradation of thepeptide both in vivo and in vitro. Alternatively,formation of the leader peptide may be pre-

,.v

PitAFIG. 7b.

vented in some manner by the secondary struc-ture of the leader RNA (10).

ACKNOWLEDGMENTSWe thank Daniel Oppenheim for help in constructing the

tip transducing phage.This work was supported by U.S. Public Health Service

grant GM 09738 from the National Institute of General Med-ical Sciences, grant PCM 73-06774-A03 from the NationalScience Foundation, and the American Heart Association.G.F.M. is a postdoctoral fellow supported by the Swiss Na-tional Foundation. C.Y. is a career investigator of the Ameri-can Heart Association.

These studies were performed in accordance with the Na-

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tional Institutes of Health guidelines on recombinant DNAresearch.

LITERATURE CITED1. Bertrand, K., C. Squires, and C. Yanofsky. 1976. Tran-

scription termination in vivo in the leader region of thetryptophan operon of Escherichia coli. J. Mol. Biol.103:319-337.

2. Bonner, W. M., and R. A. Laskey. 1974. A film detectionmethod for tritium-labeled proteins and nucleic acids inpolyacrylamide gels. Eur. J. Biochem. 46:83-88.

3. Bronson, M. J., C. Squires, and C. Yanofsky. 1973.Nucleotide sequences from tryptophan messenger RNAof Escherichia coli: the sequence corresponding to theamino-terminal region of the first polypeptide specifiedby the operon. Proc. Natl. Acad. Sci. U.S.A.70:2335-2339.

4. Files, J. G., K. Weber, and J. H. Miller. 1974. Trans-lational reinitiation: reinitiation of lac repressor frag-ments at three internal sites early in the lacI gene ofEscherichia coli. Proc. Natl. Acad. Sci. U.S.A.71:667-670.

5. Helinski, D. R., V. Hershfield, D. Figurski, and R. J.Meyer. 1977. Construction and properties of plasmidcloning vehicles, p. 151-165. In R. F. Beers and E. G.Basset (ed.), Recombinant molecules: impact on scienceand society. Raven Press, New York.

6. Helinski, D. R., and C. Yanofsky. 1962. Peptide patternstudies on the wild type A protein of the tryptophansynthetase of Escherichia coli. Biochim. Biophys. Acta63:10-19.

7. Hershfield, V., H. W. Boyer, L. Chow, and D. R.Helinski. 1976. Characterization of the mini-ColElplasmid. J. Bacteriol. 126:447-453.

8. Jackson, E. N., and C. Yanofsky. 1973. The regionbetween the operator and first structural gene of thetryptophan operon of Escherichia coli may have aregulatory function. J. Mol. Biol. 76:89-101.

9. Laemmli, U. K. 1970. Cleavage of structural proteinsduring the assembly of the head of bacteriophage T4.Nature (London) 227:680-685.

10. Lee, F., and C. Yanofsky. 1977. Transcription termina-tion at the trp-attenuators of Escherichia coli andSalnonella typhimurium: RNA secondary structureand regulation of termination. Proc. Natl. Acad. Sci.U.S.A 74:4365-4369.

11. Michels, C., and D. Zipser. 1969. Mapping ofpolypeptidereinitiation sites within the ,B-galactosidase structuralgene. J. Mol. Biol. 41:341-347.

12. Murialdo, H., and L. Siminovitch. 1971. The morpho-genesis of bacteriophage lambda. III. Identification ofgenes specifying morphogenetic proteins, p. 711-723. InA. D. Hershey (ed.), The bacteriophage lambda. Cold

Spring Harbor Laboratory, Cold Spring Harbor, N.Y.13. Newton, A. 1969. Re-initiation of polypeptide synthesis

and polarity in the lac operon of Escherichia coli. J.Mol. Biol. 41:329-339.

14. Platt, T., C. Squires, and C. Yanofsky. 1976. Ribosomeprotected regions in the leader-trpE sequence of Esch-erichia coli tryptophan operon messenger RNA. J. Mol.Biol. 103:411-420.

15. Platt, T., K. Weber, D. Ganem, and J. H. Miller. 1972.Translational restarts: AUG reinitiation of the lac re-pressor fragment. Proc. Natl. Acad. Sci. U.S.A.69:897-901.

16. Ray, P., and H. Murialdo. 1975. The role of gene Nu3 inbacteriophage lambda head morphogenesis. Virology64:247-263.

17. Sarabhai, A., and S. Brenner. 1967. A mutant whichreinitiates the polypeptide chain after chain termina-tion. J. Mol. Biol. 27:145-162.

18. Schmeissner, U., D. Ganem, and J. H. Miller. 1977.Genetic studies of the lac repressor. H. Fine structuredeletion map of the lacI gene, and its correlation withthe physical map. J. Mol. Biol. 109:303-326.

19. Selker, E., K. Brown, and C. Yanofsky. 1977. Mito-mycin C-induced expression of trpA of Salnonella ty-phimurium inserted into the plasmid ColEl. J. Bacte-riol. 129:388-394.

20. Shine, J., and L Dalgarno. 1974. The 3'-terminal se-quence of Escherichia coli 16S ribosomal RNA: com-plementarity to nonsense triplets and ribosome bindingsites. Proc. Natl. Acad. Sci. U.S.A. 71:1342-1346.

21. Squires, C., F. Lee, K. Bertrand, C. L. Squires, M. J.Bronson, and C. Yanofsky. 1976. Nucleotide se-quence of the 5' end of tryptophan messenger RNA ofEscherichia coli. J. Mol. Biol. 103:351-381.

22. Thomas, M., and R. W. Davis. 1975. Studies on thecleavage of bacteriophage lambda DNA with EcoRIrestriction endonuclease. J. Mol. Biol. 91:315-328.

23. Weber, K., and M. Osborn. 1969. The reliability ofmolecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem.244:4406-4412.

24. Yanofsky, C., V. Horn, M. Bonner, and S. Sta-siewsky. 1971. Polarity and enzyme functions in mu-tants of the first three genes of the tryptophan operonof Escherichia coli. Genetics 69:409-433.

25. Zalkin, H., C. Yanofsky, and C. L Squires. 1974.Regulated in vitro synthesis of Escherichia coli tryp-tophan operon messenger ribonucleic acid and enzymes.J. Biol. Chem. 249:465-475.

26. Zipser, D., S. Zabell, J. Rothman, T. Grodzicker, andM. Wenk. 1970. Fine structure of the gradient of polar-ity in the Z gene of the lac operon of Escherichia coli.J. Mol. Biol. 49:251-254.

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