attenuation regulation escherichia k-12: molecularcloning...

Vol. 152, No. 1JOURNAL OF BACTERIOLOGY, OCt. 1982, p. 363-3710021-9193/82/100363-09$02.0O/0Copyright 0 1982, American Society for Microbiology

Attenuation Regulation in the thr Operon of Escherichia coliK-12: Molecular Cloning and Transcription of the Controlling

RegionSTEVEN P. LYNN,1 JEFFREY F. GARDNER,'* AND WILLIAM S. REZNIKOFF2

Department of Microbiology, University ofIllinois, Urbana, Illinois 61801,1 and Department ofBiochemistry,University of Wisconsin, Madison, Wisconsin S37062

Received 9 March 1982/Accepted 17 June 1982

Recombinant plasmids were constructed which carry defined regions of thethreonine (thr) operon regulatory region of Escherichia coli. In vitro transcriptionexperiments utilizing plasmid or restriction fragment templates showed that twomajor RNA transcripts, which differ in length by one to a few bases, aretranscribed from this region. The approximate length of the transcripts is 150 to170 bases, and the site(s) of termination is near or within the thr attenuator. Theefficiency of termination at the thr operon attenuator in vitro is approximately90%o. A regulatory mutation, thr79-20, which is a G-C insertion in the attenuator,reduces the frequency of transcription termination to 75%. In addition, in vivoRNA transcripts were identified which hybridize to the thr operon regulatoryregion. These transcripts appeared to be identical to the two major in vitrotranscripts as judged by their mobilities on 8% polyacrylamide-8 M urea gels.This result indicates that the thr operon regulatory region is transcribed in vivoand that termination occurs near or within the thr attenuator.

The threonine (thr) operon of Escherichia colispecifies four of the five enzymatic activitiesnecessary to synthesize threonine from asparticacid (15, 51) (Fig. 1). The structural genes(thrABC) of the operon (51) map at minute zeroon the standard linkage map (1). A regulatoryregion, originally identified by cis-dominant con-stitutive mutations, maps adjacent to the thrAstructural gene (19, 43, 51). We were interestedin genetic regulation of the thr operon sinceexpression of the structural genes is multivalent-ly regulated by the intracellular levels of boththreonine and isoleucine (15).DNA sequencing studies have revealed that

the thr operon may be regulated by an attenua-tion mechanism (17) similar to other amino acidbiosynthetic operons (3, 12, 20, 24, 26, 27, 28,29, 38, 39, 54). Structural features of the thrregulatory region which have been identified byDNA sequence analysis include a potential cod-ing region for a "leader peptide" and a tran-scription termination site (attenuator) approxi-mately 30 base pairs (bp) preceding the thrAgene. The putative leader peptide is 21 aminoacids in length and contains eight threonine andfour isoleucine codons. The attenuator was iden-tified by its homology with other transcriptionterminators and by regulatory mutations whichresult in constitutive synthesis of the thr operonenzymes. Presumably transcription termination

at the thr attenuator is defective in these mutantstrains (17; S. Lynn, C. Bauer, K. Chapman,and J. Gardner, manuscript in preparation). Themodel for regulation of the thr operon proposesthat translation of the leader RNA is involved inregulation of transcription termination at the thrattenuator (17, 53).

This report describes the molecular cloning ofthe thr operon regulatory region and in vitrotranscription experiments localizing the thr op-eron promoter and attenuator. The in vitro tran-scription results show that transcripts approxi-mately 150 to 170 bases in length are initiated inthe thr regulatory region and terminated at ornear the attenuator. Evidence is also presentedwhich indicates that these transcripts are syn-thesized in vivo.

MATERIALS AND METHODSBacteria, bacteriophages, plasmids, and media. E.

coli K-12 strains C600 SF8 (thrB leu thi str hsr hsmrecB recC lop) (49) and MO (F-, isogenic with Hfr H)(18) were used throughout this study.

A pthr spi and A pthr79-20 spi are recombinantphages which carry the thr operon controlling ele-ments and the thrA and B structural genes (18). Apthr79-20 spi carries the constitutive thr79-20 muta-tion. Plasmids pBR322 (7) and pVH51 (22) were usedas cloning vehicles.M9, TYE, LB (37), and Thr (18) were used as base

media. The common L-amino acids (40 ,ug/ml), deoxy-

363

364 LYNN, GARDNER, AND REZNIKOFF

asportotesemialdehyde homoserine homoserine threonine

asportokinose I dehydrogenose dehydrogenose I kinose synthase(EC 2.7.2.4) (EC 1.2.1.11) (EC 1.1.1.3) (EC 2.7.1.39) (EC 2.9.9.2)

asport ic __ -osportyl osportic homoserine homoserine threonineacid phosphote 8-semioldehyde 0-phosphote

thrA asd MthrA thrB thrCII

lysine methionine isoleucine

FIG. 1. Biosynthetic pathway for L-threonine. Aspartate semialdehyde dehydrogenase (asd) is unlinked tothe thr operon and is regulated by lysine, methionine, and threonine.

cholate (0.5% wt/vol), ampicillin (50 ,ug/mi), and tetra-cycline (20 ag/ml) were used when required.Phage growth, pamid purification, and r n

fragment isolation. Bacteriophages were propagated inMO cells and the DNA was isolated as described byGardner and Reznikoff (18). Plasmid DNA was isolat-ed as described by Maquat and Reznikoff (33).

Restriction fragment templates were isolated by themethod ofMaxam and Gilbert (35) and further purifiedby DE52 chromatography as described by Maquat etal. (34). DNA samples were stored in DNA buffer (10mM Tris-hydrochloride [pH 7.91-0.1 mM EDTA) at40C.

In vitro Urnsciption conditions and DNA sequenc-ing. Reactions were incubated for 10 min at 37°C in 50,ul of 20 mM Tris-hydrochloride (pH 7.9)-0.1 mMEDTA-0.1 mM dithiothreitol-150 mM KC1-4 mMMgCI2 with 120 mM of the four unlabeled nucleosidetriphosphates, 10 to 50 gCi of [a-32P]CTP or [a-32P]UTP, and 1 to 2 gig of plasmid DNA (0.2 to 0.4pmol) or 2 to 5 pmol of restriction fiagment DNA (14).Reactions were initiated by addition of 2 gug (1 pmol) ofRNA polymerase. After 20 min, rifampin was added to10 gig/ml, and the reactions were incubated for anadditional 10 min. tRNA (25 gag) was added, and thesamples were then phenol extracted once, and 200 gulof 0.3 M sodium acetate was added to each tube. Thesamples were then ethanol precipitated at -70°C for 5min, and the pellets were washed with 95% ethanol,dried, and subjected to gel electrophoresis on 8%acrylamide-8 M urea gels as described by Maxam andGilbert (35). Quantitation of radioactivity in gel sliceswas done by Cerenkov radiation in a liquid scintilla-tion counter. DNA sequencing and 5' end labelingwere as described by Maxam and Gilbert (35). [y-32P]ATP was prepared by the method of Johnson andWalseth (23).Coddn El, r n endonucleases, enzymes, and

gel eectrophoreis conditions. Colicin El was preparedas described by Maquat and Reznikoff (33). AluI,EcoRI, HaeIII, HhaI, HindII, HindIII, RsaI, and TaqIwere prepared by standard methods (21, 32, 36, 40, 41,44, 46). BamHI, BstEII, ClaI, and Sail were pur-chased from Bethesda Research Laboratories andwere used as described by the manufacturer. T4 DNAligase and T4 DNA polymerase were purchased fromNew England Bio-Labs or purified in our laboratoryby an unpublished procedure (R. Gumport, personalcommunication). T4 polynucleotide kinase was a gen-erous gift from D. Soltis and 0. Uhlenbeck, Universityof Illinois. Bacterial alkaline phosphatase was pur-chased from Worthington Diagnostics. Agarose andpolyacrylamide gel electrophoresis were carried out as

described previously (6, 45). RNA polymerase waspurified by the method of Lowe et al. (31) or pur-chased from Bethesda Research Laboratories.Chemicals. All chemicals were of reagent grade. a-

32P-labeled ribonucleoside triphosphates were pur-chased from New England Nuclear Corp. or Amer-sham Corp. 32Pi was purchased from New EnglandNuclear.

Isolatin of thr RNA synteszed in vivo. Labeling ofcellular RNA with 32Pi (20 mCi) and RNA isolationwere performed as described by Squires et al. (47).Five-microgram samples each of BamHI-digestedpBR322 and pSL108 DNAs were subjected to agarosegel (1% wt/vol) electrophoresis, transferred to nitro-cellulose filters, and hybridized with 32P-labeled RNAas described by Davis et al. (11). Elution ofRNA fromnitrocellulose filters was accomplished by adding 300,Al of 10mM Tris-hydrochloride (pH 7.9)-i mM EDTAand the nitrocellulose strip to a 1.5-mi siliconizedEppendorf tube and heating to 95°C for 5 min. Thefilter was removed, and the RNA was precipitated byaddition of 25 gag of tRNA, 35 gul of 3 M sodiumacetate, and 1 ml of absolute ethanol and kept at-70°C for S min. The mixture was centrifuged at10,000 x g for 5 min, and the RNA was vacuum driedand subjected to electrophoresis as described above.Ligtion and transformation conditios. Ligation re-

actions (10 to 20 gAl) contained 66 mM Tris-hydrochlo-ride (pH 7.9), 6.6 mM MgCl2, 66 ,uM ATP, 10 mMdithiothreitol, and 5 mM spermidine. In experimentsutilizing pVHS1 as the vector, ligation reactions werecarried out at 15C ovemight with 0.1 to 0.2 U of T4DNA ligase. In experiments utilizing pBR322 as thevector, ligation reactions were carried out at 4°Covemight with 5 U (52) of T4 DNA ligase purified inour laboratory. Transformations, using SF8, werecarried out as described by Maquat and Reznikoff (33).After transformation, the mixture was added to 4 ml ofLB broth, and the cells were grown with shaking for 90min at 37C before colicin El treatment or directplating onto antibiotic selection plates.

Constrction of recombinant pIds. pJG10 andpJG39 contain the HaeIII 1,700-bp (HaeIII-1,700) re-striction frament from A pthr spi (18) inserted into theunique HindII site of pVHS1. Ligation mixtures con-tained 0.2 gg of HindII-digested pVHS1 and 0.1 gg ofpurified HaeIlI-1,700 DNA from A pthr spi. Afterligation, SF8 cells were transformed as describedabove, and colicin El-immune transformants wereselected. Colonies were purified and screened forplasmids by a modification of the toothpick assay (2).Approximately 10%o of the clones carried plasmidslarger than pVHS1, and comparison of the mobilities

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TRANSCRIPTION OF thr REGULATORY REGION 365

of the larger plasmids with molecular-weight standardsindicated that the recombinant plasmids were approxi-mately 1,700 bp larger than pVH51. The plasmidscarrying the HaeIII-1,700 fragment, pJG10 and pJG39,were used in subsequent studies.The structures of these plasmids (see Fig. 3) was

confirmed by restriction endonuclease analysis ofpVH51, pJG10, pJG39, and the HaeIII-1,700 fragmentDNAs with TaqI. The TaqI sites in the HaeIII-1,700fragment have been mapped previously (18). All TaqIfragments, which are derived from the internal regionsof the HaeIII-1,700 fragment, are present in digests ofpJG1O and pJG39 (Fig. 2, panel 1). The two terminalTaqI fragments are not found in digests of theseplasmids but appear as unique fusion fragments (whichrepresent fused pVH51 and HaeIII-1,700 DNA) in thedigests. Since the sizes of the fusion fragments inpJG39 and pJG10 are different, we conclude thatpJG39 and pJG10 represent opposite orientations ofthe HaeIII-1,700 fragment in the pVH51 HindII site.Experiments with HindII, HhaI, AIuI, and RsaIshowed similar results.pSL102 (Fig. 2, panel 2; Fig. 3) was constructed by

inserting a HindII-EcoRI fragment from A pthr spi intothe unique HindIII and EcoRI sites of pBR322.HindIII-EcoRI double digests of pBR322 (0.1 Fg) andA pthr spi (0.3 ,ug) were mixed and ligated. Aftertransformation, cells were plated on Thr ampicillin

A B C D2

plates to select Thr+ transformants. Plasmid DNAfrom several transformants was screened by the proce-dure of Birnboim and Doly (5), and all contained theHindIII-EcoRI fragment from A pthr spi, which hasbeen shown previously to carry thr operon DNAincluding the controlling elements, the thrA and thrBgenes, and part of thrC (18) (Fig. 3). A representativeplasmid, pSL102, was used in further constructionsand in transcription studies.pSL105 was constructed by removing the 260-bp

HindIII-BstEII fragment of pSL102 (Fig. 2, panel 2;Fig. 3). pSL102 was digested with a combination ofHindIIl and BstEII, and the recessed 3' ends werepolymerized to flush ends with T4 DNA polymeraseand the four deoxynucleoside triphosphates (30). Afterligation and transformation (see above), cells wereplated on TYE ampicillin plates to select Apr trans-formants. All Apr transformants characterized wereThr+, and direct DNA sequencing of the HindIll-BstEII junction of one of the plasmids, pSL105, dem-onstrated that flush-end ligation had occurred.pSL108 was constructed by inserting the 574-bp

TaqI fragment of pSL105 into the unique ClaI site ofpBR322. The TaqI-574 fragment is defined by a TaqIsite in pBR322 to the left of the HindIII-BstEII junc-tion of pSL105 (see Fig. 3) and the TaqI site whichimmediately precedes the thrA gene and includes theputative thr attenuator region (17) (Fig. 2, panel 2; Fig.

A 8 C D E A B C____________ ____ ____ __ 3

ab----b

120 --

70-

FIG. 2. Restriction analysis of recombinant plasmids. (Panel 1) Digestion of pJG10 and pJG39 with TaqI.Samples were subjected to electrophoresis on a 5% polyacrylamide gel. Lane A, pVH51; B, pJG10; C, HaeIII-1,700 fragment; and D, pJG39. Fragment a is the pVH51 fragment which carries the HindIl site used forconstruction of pJG10 and pJG39. Fragments b and b' and c and c' are unique to pJG10 and pJG39, respectively,and carry pVH5lHaeIII-1,700 fragment fusion DNA. The other two TaqI fragments (120 and 70) have beenmapped at the termini of the HaeIII-1,700 fragment (18). (Panel 2) Digestion of pBR322 derivatives with TaqI.Samples were subjected to electrophoresis on a 5% polyacrylamide gel. Lane A, pBR322; B, pSL108; C,pSLlO5; D, pSL102; and E, HaeIII digest of pBR322. Fragments a (394 bp) and b (440 bp) in lane D are TaqIfragments containing the HindIII site and BstEII site, respectively. ab (in lane B and C) is the HindIII-BstIIdeletion fragment (574 bp) obtained as a consequence of the construction of pSL105. The length of the HaeIIIfragments from pBR322 are as determined by Sutcliffe (50) from top to bottom: 587, 540, 504, 458, 434, 267, 234,213, 192, 184, 124, 123, 104, 89, and 80 bp. (Panel 3) Digestion of pSL123 and pSL130 with EcoRI. Samples weresubjected to electrophoresis on a 5% polyacrylamide gel. Lane A, HaeIII digest of pBR322; B, pSL123; and C,pSL130. The EcoRI-367 (AluI-HaeIII-367) fragments are indicated. The lengths of the HaeIII fragments ofpBR322 are the same as those for panel 2.

VOL. 152, 1982

b


3). Ligation conditions were as described above, using0.3 Lg of purified TaqI-574 DNA and 0.1 pg of ClaI-digested pBR322 DNA which had been treated withbacterial alkaline phosphatase. Transformed cellswere plated on TYE ampicillin plates and Apr, Tcstransformants were analyzed further by the procedureof Birnboim and Doly (5). One larger plasmid, pSL108,was used in further experiments. Insertion of a TaqIfragment into a ClaI site restores the TaqI sites; Fig. 2,panel 2 shows that pSL108 contains the TaqI-574fiagment of pSL1O5 which carries the HindII-BstIIfusion.pSL123 and pSL130 contain the 367-bp AluI-HaellI

fragments (-267 to +100; Fig. 2, panel 2; Fig. 3) fromeither A pthr spi (pSL123) or A pthr79-20 spi (pSL130),which were inserted into the EcoRI site of pBR322.Before ligation, the EcoRI ends of pBR322 werepolymerized to flush ends (30) so that the plasmid-fragment junctions would regenerate EcoRI sites. Af-ter transformation, Apr transformants were isolated,and plasmid DNA from several transformants wasscreened by the procedure of Birnboim and Doly (5).Both pSL123 and pSL130 yield a fragment, identical insize to the AluI-HaeIIl-367 fragment, when digestedwith EcoRI (Fig. 2, panel 3).

RESULTSConstruction of recombinant plasmids. Recom-

binant plasmids were constructed as described

above, and their structures are shown in Fig. 3.Recent DNA sequencing studies on the thr oper-on-controlling region (17) showed that theHaeIII-1,700 restriction fragment, carried inpJG10 and pJG39, contains the coding sequencefor the amino terminus of the thrA gene, thesecondary A attachment site (9), the attenuatorpreceding thrA, and the coding region for theleader peptide. DNA sequencing of the constitu-tive thr79-20 mutation revealed that it containedan insertion of a G-C base pair in the thrattenuator (17). The amino terminus of thrA, theattenuator, and the leader peptide all map at theextreme left end of the HaeIII-1,700 fragment(18).One of the objectives of this study was to

construct recombinant plasmids which could beused in in vitro transcription experiments tolocalize the position of the thr promoter and tostudy termination at the thr attenuato,. As aresult, we constructed plasmids pSL102,pSL105, pSL108, pSL123, and pSL130 (Fig. 3).pSL102 and pSL105 carry thr regulatory DNAdefined by the Hindlll and the BstEII sites,respectively, and carry the thr attenuator andstructural gene DNA extending to the EcoRI site

pSL102

Kpn

EtoRI

pJG 39

pJG 10

tj tht C'

Kpn, EcoRI

tht2AI

thrt A, 1T thr L

RN TGq R" R"

100 25 1 -25 -71 -76 - 15 -154Hind EIIL

Sol s

+ Hitd

THin-254-267

Tcq

"he -H-*III 264 f.sgssst

h5o Al 367 frms

Bt LI1-eossI-354 f-,5-st

Set 5 11 - Cie I - 377 fr_pmt

EcoRl EcoRIE E

E10oRISetEll* $

FIG. 3. Restriction map of the thr operon regulatory region and recombinant plasmids. A partial map of thethr operon regulatory region has been published (18), and the other restriction sites were deduced from the DNAsequence (17). The thr operon attenuator region is designated thrT and is located between the RsaI site at -76and the TaqI site at -25. The plasniids pJG10, pJG39, pSL102, pSL1O5, pSL108, and pSL123 were constructedas described in the text. The EcoRI-367, HhaI-HaeIII-284, BstEII-EcoRI-354, and BstEII-ClaI-377 fragments are

shown. =, vector sequences; em, A sequences; -, E. coli chromosomal sequences.

3010

p5L WS

p8L 123 or pSL130

v v-3

v=:t.L

J. BACTERIOL.

EcoRl4

PSL 105

EcoRl Hind III

EcOR

---C±==:

Kpn

-C=.=

th, 8

H"lill lS Ell Hind Ill Hz IIl

tht P

Set Ell

The Hhs Ahu

I-456 - 5t4

Eco REco RI

=1f

cisKft 01

Eco RI

Nhl

cis lht Ell


in thrC. pSL108 carries thr regulatory DNAextending from the BstEII site (-254) to theTaqI site (-25). This plasmid carries the thrattenuator in the opposite orientation than thatin pSL102 and pSL105 but does not include anystructural gene DNA (data not shown). pSL123and pSL130 carry thr regulatory DNA definedby the AluI (-267) and HaeIII (+100) sites,respectively. These plasmids contain the attenu-ator and the coding sequence for the first 33amino acids of the thrA gene. pSL130 carries theconstitutive thr79-20 mutation from X pthr79-20spi.

In vitro transcription studies. The recombinantplasmids described in the previous section andpurified EcoRI-367 (Alul-HaeIII-367) restrictionfragment DNA from pSL123 were used as tem-plates in initial in vitro transcription studies.RNA was synthesized as described above byusing [a-32P]UTP as the label. Figure 4 shows anautoradiograph of an 8% polyacrylamide-8 Murea gel. The results show that two major dis-crete RNA products, almost identical in size, aretranscribed from the recombinant plasmids andthe EcoRI-367 fragment but not from pVH51 orpBR322 templates. Transcription with the HhaI-HaeIII-284 fragment as template does not directthe synthesis of the RNAs (data not shown). Themobility of the RNAs, relative to the 6S P'Rtranscript from bacteriophage X is consistentwith a size of approximately 150 to 170 bases(data not shown).

Transcription from the EcoRI-367 fragmentisolated from pSL130, which carries the thr79-20attenuator mutation, yields slightly different re-sults (Fig. 5, lanes A and B). The transcriptscorresponding to the terminated RNA migratemore slowly than the wild-type transcripts on8% polyacrylamide-8 M urea gels (Fig. 5, lanesC and D). The difference in mobility is greaterthan expected for the one-base-pair insertion inthe thr79-20 mutation. Since the template usedin this experiment carries the thr79-20 mutation,the difference in the mobility of the mutanttranscript may be due to the difference in sec-ondary structure at the 3' end of the transcript,so that the mobility of the RNA on a polyacryl-amide-urea gel is different from that of the wild-type RNA (8). Alternatively, the presence of thethr attenuator mutation could change the termi-nation site so that the transcripts are longer thanwild-type transcripts.

Efficiency of transcription termination at thethr operon attenuator in vitro. Experiments withpurified restriction fragment templates were per-formed to measure the frequency of transcrip-tion termination at the thr attenuator. To quanti-tate transcription termination, it is necessary tomeasure both the terminated RNA and the "run-off' RNA which is transcribed to the end of the

A B c D E F G H

FIG. 4. Autoradiograph of in vitro transcriptionreactions from recombinant plasmids. In vitro tran-scriptions were performed as described in the text, andsubjected to electrophoresis on an 8% polyacryl-amide-8 M urea gel. Lane A, pJG10; B, pBR322; C,pSL108; D, pSL105; E, pSL102; F, EcoRI-367 frag-ment; G, pSL123; H, pVH51; and I, pJG39. Theposition of the terminated transcripts are indicated bythe arrow. It can be seen that these RNAs are the onlycommon species transcribed from the recombinantplasmids and the EcoRI-367 fragment which carriesthe thr operon attenuator region.

restriction fragment template. The templatesused in these experiments were the BstEII-EcoRI-354, EcoRI-367, and BstEII-ClaI-377fragments from pSL123 (Fig. 3). The read-through transcripts from the EcoRI-367 andBstEII-EcoRI-354 templates should be identicalin length since the same EcoRl site, distal to theattenuator, was used to generate the fragments.However, the runoffRNA from the BstEII-Clal-377 fragment should be approximately 23 baseslonger since the ClaI site is 23 bases further fromthe attenuator. The results (Fig. 6) show thatlonger transcripts from the EcoRI-367 and theBstEmi-EcoRI-354 fragments are identical inlength and are slightly smaller than the transcript

VOL. 152, 1982


attenuator. The efficiency of transcription termi-nation when the in vitro transcription system isprogrammed with the EcoRI-367 fragment frompSL130, which contains the thr79-20 constitu-tive mutation, is approximately 75%.Trnmription of the thr regulatory region in

vivo. If transcription initiation and termination invivo is similar to that observed in vitro, onewould predict that it might be possible to isolatea chromosomal RNA transcript, similar in sizeto the in vitro transcript, by hybridizing total

X;

FIG. 5. Autoradiograph of in vitro [a-32P]UTP-la-beled transcripts from wild-type (thr79-2) and thr79-20EcoRI-367 fragment templates. Samples were subject-ed to electrophoresis in an 8% polyacrylamide-8 Murea gel. Lanes a and b contain the thr79-20 EcoRI-367fragment and lanes c and d contain wild-type (thr79-2)EcoRI-367 fragment. Lanes a and c display tran-scriptions performed with 30 ,Ci of [a-32P]UTP, andlanes b and d show transcriptions performed with 10,uCi of [Q-32P]UTP. The faster-migrating species ineach lane are the terminated RNAs, and the slower-migrating species represent runoff transcripts from thefragment templates.

from the BstEII-ClaI-377 fragment. The resultsof several experiments with the wild-typeEcoRI-367 fragment as template and [a-32P]CTPor [a-32P]UTP as the label for in vitro transcrip-tion are summarized in Table 1. After convertingfor the base composition of the label in thetranscripts, these data indicate that terminationis approximately 90%o efficient at the wild-type

FIG. 6. Autoradiograph of in vitro transcripts la-beled with [a-32P]CTP from templates carrying varioussegments of the wild-type thr operon regulatory re-

gion. Samples were subjected to electrophoresis in an8% polyacrylamide-8 M urea gel. Lane a, EcoRI-367fragment; b, BstEII-EcoRI-354 fragment; and c and d,BstEll-ClaI-377 fragnent. The terminated RNAs arethe faster-migrating species in each lane. The runofftranscripts are the slower-migrating species. The run-

off transcripts increase in length when the attenuatordistal end of the fragment template increases in length.

. ...

J. BACTERIOL.


TABLE 1. Frequency of transcription termination atthe thr attenuator

[a-32Pribo- TerminationTemplate nucleoside fencyo

triphosphatea frequency

Wild-type EcoRI-367 CTP 92 ± 2Wild-type EcoRI-367 UTP 90 ± 3thr79-20 EcoRI-367 CTP 78thr79-20 EcoRI-367 UTP 75

a Radiolabeled nucleoside triphosphate used in thein vitro transcription reaction.

b Frequency was calculated as terminated RNAI(terminated RNA + readthrough RNA * X) where X isthe correction factor for label distribution assuminginitiation at position -190 (Fig. 3). If it is assumed thatthe initiation is between -200 and -180 based on thesize of the RNA, the error for CTP labeling is 1 to 1.5%and for UTP is 1 to 2%.

cellular 32P-labeled RNA to plasmids carryingthe thr regulatory region. Strain MO was grownin the presence of 32P1, and RNA was extracted,hybridized to nitrocellulose filters containingdenatured linear pBR322 or pSL108 DNA, andeluted from the filters as described above. Fig-ure 7 shows an 8% polyacrylamide-8 M urea gelcontaining RNA eluted from pBR322 andpSL108. In addition, RNA transcribed in vitrofrom pSL108 was subjected to electrophoresis inan adjacent lane. The results show that twodiscrete in vivo RNA species, identical in size tothe two major in vitro transcripts, hybridize topSL108 DNA but not to pBR322 DNA. Inaddition, several smaller species of RNA, whichmay represent degradation products, also hy-bridize to pSL108 DNA.

DISCUSSIONIn previous studies, the DNA sequence of the

thr operon regulatory region carried on theHaeIII-1,700 fiagment (17, 18) was mapped anddetermined. DNA sequence analysis of the regu-latory region indicated that its organization issimilar to the regulatory regions of the trp (29,39), phe (54), his (3, 12), leu (20, 27), and ilv (28,38) operons, which appear to be regulated by anattenuation mechanism. The organization issuch that the promoter region is proximal to apotential coding region for a regulatory peptide,which is followed by an attenuator site. Theattenuator site, which precedes the structuralgenes of the operon, is a site where RNApolymerase either terminates transcription orproceeds into the structural genes, dependingupon the availability of the regulatory aminoacid(s).The thr attenuator is structurally similar to

several other "rho-independent" terminators inthat it contains a region of dyad symmetry highin G-C content, followed by an A-T-rich region

of from 4 to 9 base pairs in length (42). Recentstudies have indicated that both the RNA sec-ondary structure, encoded by the region of dyadsymmetry, and the run of uridine residues, en-coded by the A-T rich region, are necessary fortranscription termination at these terminators(10, 13, 14, 42). The constitutive thr79-20 muta-tion, which contains a G-C base pair insertion inthe region of dyad symmetry of the attenuator,disrupts the RNA secondary structure of theattenuator so that the frequency of transcriptiontermination may be reduced in vivo. Similarmutation in the trp and his attenuators have beendescribed by Stauffer et al. (48) and Johnstonand Roth (25), respectively.

A B C

FIG. 7. Autoradiograph of in vivo RNAs elutedfrom pBR322 and pSL108. The RNA samples wereobtained and processed as described in the text. LaneA, elution from pBR322; B, elution from pSL108; andC, in vitro transcription with pSL108 as template.These samples were subjected to electrophoresis onthe same 8% polyacrylamide-8 M urea gel. Autoradi-ography for the in vitro RNA was for 1 h at -20°C andfor the in vivo RNAs for 3 days at -70°C.

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The in vitro transcription experiments withboth plasmid and restriction fragment templatesshowed that two major discrete RNA species,which differ in length from one to a few bases,are transcribed from the thr operon regulatoryregion. It is not known whether this heterogene-ity occurs at the 5' or 3' or both ends of thetranscript, but examples of both are known (42).The transcripts are initiated between the BstEII(-254) and HhaI (-184) sites since the HhaI-HaeIII-284 template does not direct the synthe-sis of these RNAs. The DNA sequence near theBstEII site shows some homology to knownpromoter sequences (J. Gardner, manuscript inpreparation), and direct RNA sequencing stud-ies are required to further identify the thr pro-moter. The results from transcription of thepSL108 template indicate that transcription ter-mination occurs before the TaqI site (-25)which lies immediately distal to the stretch of A-T residues in the thr attenuator (17). The mostlikely termination site is at or near the thrattenuator itself. The region between the BstEIIand TaqI sites is 226 bp in length and is largeenough to accommodate both a promoter andthe 150- to 170-base transcript.Experiments utilizing [a-32P]UTP or

[a-32P]CTP as the in vitro label indicate that thefrequency of termination at the thr attenuator isapproximately 90%o. This is similar to the valueof95% reported for termination at the trp attenu-ator (48, 55). Interestingly, transcripts synthe-sized from templates bearing the thr79-20 muta-tion showed an altered mobility on 8%polyacrylamide-8 M urea gels, and the termina-tion frequency was reduced to 75%. The thr79-20 mutation, a G-C base pair insertion in theregion of dyad symmetry of the attenuator,disrupts the predicted RNA secondary structureof the termination structure so that the frequen-cy of transcription termination may be reducedin vivo. The thr79-20 mutation causes a fivefoldincrease in the expression of thr operon en-zymes in vivo (19), but any simple direct correla-tion of increased enzyme levels and transcrip-tional readthrough frequency is difficult to makewithout analysis of other attenuator mutations.The various models for attenuation regulation

predict that transcription termination should oc-cur at attenuators in vivo. Bertrand et al. (4)have isolated the in vivo trp-terminated leaderRNA and Frunzio et al. (16) have isolated the hisleader RNA transcribed from minicells contain-ing plasmids carrying the his leader region. Ourresults show that it is possible to isolate two invivo RNA products which are transcribed fromthe thr operon regulatory region of the E. colichromosomal DNA. The mobilities of thesetranscripts appear to be identical to those of thein vitro transcripts. These results strongly sug-

gest that the thr operon regulatory region istranscribed in vivo and that the transcripts aresimilar, if not identical, to the two major in vitrotranscripts.

ACKNOWLEDGMENTS

This work was supported by postdoctoral fellowships fromthe National Institutes of Health and the American CancerSociety (J.F.G.), grant PCM 78-07931 (J.F.G.) from the Na-tional Science Foundation, and Public Health Service grantGM19670 (W.S.R.) from the National Institute of GeneralMedical Science.

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attenuation regulation escherichia k-12: molecularcloning...

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