PLASMID 6, 112-118 (1981)Construction and Fine Mapping of Recombinant Plasmids Containingthe rrnB Ribosomal RNA Operon of E. co/iJCJRGEN BROSIUS,*~~ AXEL ULLRICH,~‘ MARY ALICE RAKER,* ALANE GRAY,?THOMAS J. DULL,*3 ROBIN R. GUTELL,* AND HARRY F. NOLLER**Thimann Laboratories, University of Californiu. Sunta Cruz, CaliJornia 95064, and Wenentech. Inc. I460 Point San Bruno Boulevard, South San Fruncisco, Culijornia 94080Received October 28, 1980: revised February 25, 1981We have constructed recombinant plasmids containing the entire Escherichiu co/irrnB ribosomal RNA operon and segments thereof. Cloning of the 7.5kb BamHI frag-ment, from Artin which contains this operon, in plasmid vectors pBR 313 or pBR 322is described. The 3.2-kb EcoRIIBamHI fragment containing the 3’ two-thirds of the 23 SrRNA gene, the SS rRNA gene, and the terminator region has been cloned separatelyin pBR 313. As the nucleotide sequences of pBR 322 and the 7.5kb fragment carrying therrnB operon have been established, the entire 11.9-kb sequence of pKK 3535 is now known.This makes possible precise rearrangements and site-specific alterations of the ribosomalRNA operon; thus, pKK 3535 becomes a powerful tool for studies such as initiationand termination of transcription, processing of rRNA precursors, and investigations ofthe structure, function, and assembly of the ribosome itself. A detailed physical mapof pKK 3535 is presented.Initially, our rationale in cloning rRNAgenes from Escherichia coli was the de-termination of the 16 S RNA and 23 S RNAprimary structures (Brosius et al., 1978,1980). Subsequently, sequences of tran-scriptional signals and spacer regions flank-ing rRNA and tRNA genes in rrnB andtheir comparison with homologous regionsof other rRNA operons sequenced in otherlaboratories have raised questions concern-ing aspects of initiation and termination oftranscription, as well as steps involved inthe processing of primary transcripts. A po-tential use of the recombinant plasmidsdescribed here, especially of pKK 3535for which we now know the entire nucleotidesequence, lies in specific alteration of func-tional sequences and subsequent in vitroand in viva analysis of the impact on: (a)the control of expression of a ribosomalRNA operon, (b) processing mechanisms for1 Present address: The Biological Laboratories, Har-vard University, 16 Divinity Ave., Cambridge, Mass.02138.rRNAs and tRNAs, and (c) the structure,function, and assembly of the ribosome.MATERIALS AND METHODSIsolation of DNAStrains harboring plasmids pBR 322, pBR313, and pTUB 2 were kindly provided byR. Rodriguez, M. Betlach, and Y. Kaziro,respectively. Plasmid DNA was preparedin CsCl-ethidium bromide buoyant densitygradients (Clewell, 1972). Bacteriophagehrifd18 was isolated from an E. cofi K-12strain by Kirschbaum and Konrad (1973).Phage DNA was a gift from R. Young.Restriction Enzyme DigestionMost of the enzymes were purchasedfrom New England Bio-Labs or fromBethesda Research Laboratories. EcoRIwas purified as described by Palmer et al.(1979). Reactions were carried out underthe conditions recommended by the suppliers.0147-619X/81/040112-07$02.00/0Copyright 0 1981 by Academic Press, Inc.AU rights of reproduction in soy form reserved.112
rRNA OPERON PLASMIDS 113Ligation, Transformation, and Selection ofRecombinant Plasmid-Containing CellsLigation was carried out on appropriatelydigested and phenol-extracted DNA underthe conditions of Sgaramella (1972) as de-scribed by Palmer et al. (1979). For the con-struction of pKK 123 we ligated 2 pg ofEcoRIIBamHI-digested pTUB 2 DNA, whichcarries the 18.6% EcoRI fragment of Xrifd18in pRSF 2124 (Miyajima et al., 1979) with1 pg of pBR 313 vector DNA cut with thesame enzymes using 1.5 units of T4 DNAligase (Bethesda Research Laboratories)at 12°C overnight. For construction of pKK2361, 2.4 pg of BamHI-cut hrifd 18 DNAwas ligated with 1 pg of BamHI-cut pBR313 DNA under the same conditions. Plas-mid pKK 3535 was obtained by mixing 1pg BamHI-cut pKK 2361 DNA with 2 kgof BamHI-cut and bacterial alkaline phos-phatase (Sigma)-treated pBR 322 DNA(Ullrich et al., 1977). After ligation the DNAwas ethanol precipitated and dissolved in 10mM Tris/HCl, pH 7.5, 5 mM MgC&, 50 mMCaCl,, and 200 ~1 cells were transformedwith 0.2 pg of DNA according to the pro-cedure of Bolivar et al. (1977b). E. colistrains RR1 (F-pro leu thi lacy Str’ r,-mk- endoII) (Bolivar et al., 1977a) andHBlOl (F- pro lea thi lacy Str’ r,- mB-endoI-, recA-) (Boyer and Roulland-Dus-soix, 1969) were used as recipients forthe recombinant plasmids.After transformation the cells were selectedon Luria broth plates, containing 20 E.Lg/mlampicillin and picked onto Luria brothplates containing 10 pg/ml tetracycline.Ampicillin-resistant and tetracycline-sensi-tive colonies were screened for recombinantplasmids of increased size (Barnes, 1977)and small amounts of plasmid DNA wereisolated according to Meagher et al. (1977)for further characterization by restrictionenzyme mapping. Fragmented DNA wasanalyzed on 1% horizontal agarose gels or6 or 8% polyacrylamide gels as describedelsewhere (Palmer et al., 1979).Preparation of Plasmid insertsInserts from recombinant plasmids wereseparated from their vector DNA by su-crose gradient centrifugation as describedby Valenzuela et al. (1977).RESULTS AND DISCUSSIONpKK 123The BamHIIEcoRI-digested DNA fromplasmid pTUB 2 (Miyajima et al., 1979)containing the 3’ two-thirds of the 23 S RNAgene on the 18.6% EcoRI fragment of Arifd 18(Lindahl et al., 1977) was ligated to pBR313 digested with the same enzymes. Wechose the larger plasmid pBR 313 (9.2 kb)over pBR 322 (4.3 kb) as vector becausethe desired 3.2-bp insert containing part ofthe 23 S RNA gene is then more easily re-solved from the vector DNA by sucrosegradient centrifugation. E. coli strain RR1was transformed. Out of 50 ampicillin-resistant colonies, 14 were tetracyclinesensitive. We isolated the DNA from 8 colo-nies in a “miniscreen” procedure (Meagheret al., 1977). Four out of eight samples,which were double digested with BamHIand EcoRI, carried the 3.2-kb fragmentand three carried fragments in the size rangeof 5-6 kb, which are probably the largerBamHIIEcoRI fragment from the 18.6%EcoRI fragment from tiz?lS carried bypTUB 2 or a BamHIIEcoRI fragment fromthe pRSF 2124 vector used for the con-struction of pTUB 2 (Miyajima et al., 1979).One plasmid, pKK 123, carries the 3.2-bpfragment, shown schematically in Fig. 1.Bernardi and Bernardi (1979) have inde-pendently constructed a plasmid (pAB 99)which carries the same 3.2-bp fragmentinserted in pBR 322.pKK 2361Because of low yields of plasmid DNAfrom strains carrying plasmid PER 24(Palmer et al., 1979), which contains thepromoter region of the rrnB operon, we at-
114 BROSIUS ET AL.0 I 2 3 4 5 6 7 6 kbpI L 1 I ,1 1I r, 41Ram HII A t tHindm Eco RI Eco RI Barn HIpER24II I IpERi pKK 123IpKK 2361, pKK 3535pKK 116FIG. 1. Schematic of the region of A$‘18 used in these studies. the orientation of the phage DNAmap is reversed from the usual convention, to show the rRNA operon in its conventional orientation.Wild-type A sequence is shown by hatching , and mature rRNA sequence is shown by black bars. Thescale shows DNA length in kilobase pairs. Sites of restriction enzyme cleavage used in cloning areshown. Open bars at the bottom show the cloned segments present in the recombinant plasmids. ThepER24 and pER18 segments were cloned in Co1 El (Palmer et al., 1979), pKKll5 in pBR322(Brosius et nl., 1978), pKK123 and pKK2361 in pBR313, and pKK3535 in pBR322 (cf. Fig. 2).The orientation of the insert in pKK2361 is analogous to that of pKK3.535. P, and P, are the twotandem rRNA promoters for rmB. T, and T, are putative transcriptional terminators (Brosius et al.,1981).tempted to clone the entire vrnB operon,which is included in the 7.5kb BumHI frag-ment of transducing phage hrifd 18 (Borosand Sain, 1977). We initially chose pBR313 as vector, because its larger size facili-tates the isolation of the desired BarnHI/EcoRI fragment (2189 bp), or the BamHIlHind111 fragment (1596 bp), carrying therrnB promoter region. After ligation of amixture of the Xrifdl8 BumHI fragmentswith pBR 313, we transformed E. coli strainHBlOl and screened colonies for plasmidsof the predicted size range (Barnes, 1977).Plasmid DNA from eight such colonies wasisolated by the “miniscreen” procedure(Meagher et al., 1977). The plasmids weredigested with Hind111 and the resultingfragments were electrophoresed on a 6%polyacrylamide gel to identify plasmids con-taining the unique 0.6-kb Hind111 fragmentlocated at the 5’ end of the 16 S RNA geneof the rrnB operon (Brosius et al., 1978).Three transformants contained the fragmentand were further tested by digestion ofplasmid DNA with EcoRI or double diges-tion with EcoRI/BumHI orHindIIIIBamH1.The resulting fragments were electrophoresedon a 1% agarose gel with size markersincluding the 2.2-kb insert from PER 18(Palmer et al., 1979), and the 3.2-kb insertfrom pKK 123 (not shown). Plasmid DNAfrom the tested colonies gave rise to thepredicted fragments, indicating that all threecontain the 7.5kb BumHI fragment fromkifd18, carrying the entire rrnB operon inthe same orientation with respect to thevector.One of the colonies (containing plasmidpKK 2361) was grown in supplementedM9-glucose medium (Bolle et al., 1968).We obtain this plasmid in a yield of about2 mg/liter. There is no indication of segrega-tion of the plasmid as in the case of pER24 (Palmer et al., 1979).
SacIISac II 1000:kSac IIBal IXma III115rRNA OPERON PLASMIDSPst ITth 111 IFIG. 2. Schematic map of hybrid plasmid pKK 3535. Positions of vector DNA or inserts from otherplasmids carrying parts of the rrnB operon are indicated on the inner circle. The A portion of the7.5kb fragment is hatched. The genes for the rRNAs and tRNA2’” are represented by filled bars. Twoopen reading frames (ORF I and ORF II) flanking the rrnB operon are indicated. The tandem rRNApromoters PI and P2 and their sites of initiation of transcription are indicated by arrows. A putativepromoter proximal to ORF II is indicated as PORFii. Putative terminators for the rrnB operon aremarked as Tl and T2. The ampicillin and tetracycline genes (the latter is interrupted by the 7.5-kbBamHI insert) of plasmid vector pBR 322 are dotted. The direction of transcription is indicatedby arrows under the genes. The location of these landmarks and the location of restrictionenzyme (those which recognize a sequence of six nucleotides) sites are based on the known sequenceof pKK 3535 via the primary structures of pBR 322 (Sutcliffe, 1978a) and the 7.5-kb insert (Brosiuset al., 1981). Locations of all but the sites AvrII, &/I, BclI, ClaI, SphI, Trh 1111, and XmaIII havebeen confirmed by digestion of pKK 3535 with various enzymes (see Fig. 2; Table 1).The identity of the plasmid insert was parently identical to pKK 2361 (also withfurther confirmed by restriction mapping respect to the orientation of the insert).with restriction enzymes recognizing se-quences of four or five nucleotides and byDNA sequencing (Brosius et al., 1981).pKK 3535Kiss et al. (1978) have independently As the nucleotide sequence of both theconstructed a plasmid (2/12) which is ap- 7.5kb insert of pKK 2361 and the 4.3-kb
116 BROSIUS ET AL.plasmid pBR 322 have recently becomeavailable (Brosius et al., 1981; Sutcliffe,1978a) it was desirable to subclone the 7.5kb BarnHI fragment from pKK 2361 intotheBamH1 site of pBR 322. After ligation E.coli strain HBlOl was transformed andampicillin-resistant, tetracycline-sensitivecolonies were screened for plasmids withthe predicted size (Barnes, 1977). Threeout of twelve transformants were furthertested by digestion of their plasmid DNAwith EcoRI. In all cases three fragments(6.2, 3.5, and 2.2 kb) were resolved on a1% agarose gel, indicating that the 7.5kbfragment carrying the rrnB operon wascloned into vector pBR 322 in the sameorientation as in pKK 2361 (see Fig. 2 forthe detailed physical map of pKK 3535).Cells from one of the three positive colonieswere grown and plasmid pKK 3535 was iso-abcdefghi jk lmnopqFIG. 3. Restriction patterns resolved on a 1% agarosegel of pKK 3535 digested with various enzymes:Lanes (a) BarnHI; (b) uncut pKK 3535 (c) Egll; (d)BglII: (e) BstEII; (f) EcoRI: (g) HindIII; (h) HpI:(i) PstI; (j) PvuI; (k) PruII: (1) SalI: (m) SmuI: (n)WI: (0) SacII; (p) XbaI; (q) hDNA digested withHindIII. The patterns in lanes (a) and(k) reveal in addi-tion to the expected fragments linear pKK 3535 DNAdue to incomplete digestion. The smear in lane (m) iscaused by exonuclease activity present in the SmaIpreparation. The larger of the expected bands dis-appeared completely, while the smaller 0.77-kb frag-ment is still visible. Lane (c) shows the expectedrestriction pattern of A DNA cut with HindIII; theadditional band above the 23.7-kb band and the lowyield of the 4.3-kb band is due to the cohesive ends ofADNA. Fragments smaller than 0.5 kb are not visible.TABLE 1SIZES OF FRAGMENTS GENERATED BY DIGESTIONOF pKK3535 DNA WITH VARIOUSRESTRICTION ENZYMES”Fragment sizeW)BamHI 7.5, 4.4BglI 5.0, 2.9, 2.3, 1.4 (0.2)BglII 11.9Bst EII 11.9EcoRI 6.2, 3.5, 2.2Hind111 5.7, 5.6, 0.6HpaI 11.9PSI 8.3, 3.5PVUI 11.9PVUII 7.0, 4.9SulI 6.8, 2.6, 2.5 (0.1)SmClI 11.0, 0.8SstI 11.9Sac11 6.8, 3.5, 1.4 (0.2)XbaI 11.9Hind111 (ADNA) 23.7,9.5,6.7,4.3,2.3,2.0(0.6)(0.1)fl Numbers in parentheses represent predicted frag-ments not visible on the gel (Fig. 3) due to small size.lated. The yield of pKK 3535 is about 0.2mg/liter, much lower than that of pKK 2361.However we do not observe segregationof the plasmid. The basis for the differ-ence in yield between pKK 2361 and pKK3535 is unknown.DNA from plasmid pKK 3535 was furtheranalyzed by digestion with BarnHI, Bgll,BglII, BstEII, EcoRI, HindIII, HpaI, PstI,PvuI, PvuII, SacII, SalI, SstI, and XbaIon a 1% agarose gel (Fig. 3). The resultingfragments are summarized in Table 1 andare in complete agreement with the nucleo-tide sequence (11,864 bp) of pKK 3535.The 7.5-kb insert of pKK 3535 and varioussubfragments thereof (isolated in part fromother described plasmids) have been ex-tensively mapped with about 35 restrictionenzymes, mainly as a prerequisite for gen-erating fragments for DNA sequence de-termination. The results are in accord withthe nucleotide sequence of the 7.5-kb insert(Brosius et al., 198l), except where the
rRNA OPERON PLASMIDS 117digest pattern indicates that predicted sitesremain uncut. These sites include in the7508bp fragment: (a) HphI at positions3049, 4180, and 7098; (b) TaqI at position5385; and (c) Sau96I and AvaII at position7419. In cases (a) and (b) an MboI site(G-,A-T-C) overlaps the HphI sites (G-G-T-G-A or T-C-A-C-C) or the TuqIsite (T-C-G-A) thus including the methyl-ated A residue in the HphI or TuqI recog-nition site. This is a likely reason for thefailure of these enzymes to cut if the DNA,as in our case, is isolated from a dum+,dcm+strain. Digestion at a much slower rate wasalso observed at the TuqI site at position1125 of pBR 322 (G. Sutcliffe, personal com-munication), which is similarly overlappedby an MboI site. In case (c) an EcoRII site(C-,C-$-G-G) overlaps the Suu961 orAvuII site (G-G-N-C-C/G-G-$-C-C)including the methylated C residue in theirrecognition site. It has been described pre-viously (Sutcliffe and Church, 1978) that theAvuII site at position 1438 of pBR 322 isdigested at a rate about 10 times slowerthan the remaining AvuII sites. The Suu961site at position 3247 of the 7.5-kb fragment,which is also overlapped by an EcoRII site,has not yet been tested by enzymaticdigestion.For an extensive restriction map of pBR322 see Sutcliffe (1978b). All restriction sitesoccurring in pKK 3535, including the 7.5-kb fragment carrying the rmB operon, havebeen compiled and are available from theauthors on request.REFERENCESBARNES, W. J. (1977). Plasmid detection and sizingin single colony lysates. Science 195, 393-394.BERNARDI, A., AND BERNARDI, F. (1979). Construc-tion in vitro of hybrid plasmids carrying all theEco Rl fragments from A,+fdlS DNA. Eur. J. Bio-them. 95, 391-398.BOLIVAR, F., RODRIGUEZ, R. L., BETLACH, M. C.,AND BOYER, H. W. (1977a). Construction andcharacterization of new cloning vehicles. I. Ampicil-lin-resistant derivatives of the plasmid pMB9. Genr2, 75-93.BOLIVAR, F., RODRIGUEZ, R. L., GREENE, P. J.,BETLACH, M. C., HEYNEKER, H. L., BOYER,H. W.,CROSA, J. H., AND FALKOW, S. (1977b). Con-struction and characterization of new cloning vehi-cles. II. A multipurpose cloning system. Gene 2,95-113.BOLLE, A., EPSTEIN, R. H., SALSER, W., ANDGEIDUSCHEK, E. P. (1968). Transcription duringbacteriophage T4 development: Synthesis and rela-tive stability of early and late RNA. J. Mol.Biol. 31, 325-348.BOROS, I., AND SAIN, B. (1977). Restriction endo-nuclease analysis of the transducing bacteriophagehriJn18. Mol. Biol. Rep. 3, 451-457.BOYER, H. W., AND ROULLAND-DUSSOIX, D. (1%9).A complementation analysis of the restriction andmodification of DNA in Escherichia coli. J. Mol.Biol. 41, 459-472.BROSIUS, J., PALMER, M. L., KENNEDY, P. J., ANDNOLLER, H. F. (1978). Complete nucleotide se-quence of a 16s ribosomal RNA gene from Escheri-rhia coli. Proc. Nat. Acad. Sci. USA 75, 4801-4805.BROSIUS, J., DULL, T.. AND NOLLER, H. F. (1980).Complete nucleotide sequence of a 23s ribosomalRNA gene from Escherichia coli. Proc. Nat. Acad.SC;. USA 77, 201-204.BROSIUS, J., DULL, T., SLEETER, D. D., ANDNOLLER, H. F. (1981). Gene organization and pri-mary structure of a ribosomal RNA operon fromEscherichia coli. J. Mol. Biol. 148, 107-127.CLEWELL, D. B. (1972). Nature of Co1 El plasmidreplication in Escherichia coli in the presence ofchloramphenicol. J. bacterial. 110, 667-676.KIRSCHBAUM, J. B., AND KONRAD, E. B. (1973). Iso-lation of a specialized lambda transducing bacterio-phage carrying the beta subunit gene for Escherichiacoli ribonucleic acid polymerase. J. Bacterial. 116,517-526.KISS, A., SAIN, B., KISS, I., BOROS, I., UDVARDY,A., AND VENETIANER, P. (1978). Cloning of an E.coli ribosomal RNA gene and its promoter regionfrom A$‘l8. Gene 4, 137- 152.LINDAHL,L., YAMAMOTO,M.,NOMURA,M., KIRSCH-BAUM, J. B., AI-LET, B., AND ROCHAIX, J. D. (1977).Mapping of a cluster of genes for componentsof the transcriptional and translational machineriesof Escherichia coli. J. Mol Biol. 109, 23-47.MEAGHER, R. B., TAIT, R. C., BETLACH, M., ANDBOYER, H. W. (1977). Protein Expression in E.co/i minicells by recombinant plasmids. Cell 10,521-536.MIYAJIMA, A., SHIBUY~, M., AND KAZIRO, Y. (1979).Construction and characterization of two hybrid Co1El plasmids carrying Escherichia coli trlfB gene.FEES Lett. 102, 207-210.PALMER, M. L., RAKER, M. A., KENNEDY, P. J.,
118 BROSIUS ET AL.YOUNG, J. W., BARNES, W. M., RODRIGUEZ,R. L., AND NOLLER, H. F. (1979). Isolation andrestriction mapping ofplasmids containing ribosomalDNA sequences from the rrnB cistron of E. co/i.Mol. Gen. Genet. 172, 171-178.PRIBNOW, D., SIGURDSON, D. C., GOLD, L., SINGER,B. S., BROSIUS, J., DULL, T., AND NOLLER, H. F.(1980). The rII cistrons of bacteriophage T4: DNAsequence around the intercistronic divide and posi-tions of genetic landmarks. J. Mol. Biol., in press.ROBERTS, R. J. (1980). Restriction and modificationenzymes and their recognition sequences. Gene8, 329-343.SGAMARELLA, V. (1972). Enzymatic oligomerization ofbacteriophage P22 DNA and of linear simian virus40 DNA. Proc. Nui. Acud. Sci. USA 69, 3389-3393.SLJTCLIFFE, J. G. (1978a). Complete nucleotide se-quence of the Escherichia coli plasmid pBR322.Cold Spring Harbor Symp. Quant. Biol. 43, 77-90.SUTCLIFFE, J. G. (1978b). pBR322 restriction mapderived from the DNA sequence: Accurate DNAsize markers up to 4361 nucleotide pairs long. Nucl.Acids Res. 5, 2721-2728.SUTCLIFFE, J. G., AND CHURCH, G. M. (1978).The cleavage site of the restriction endonucleaseAva II. Nucl. Acids Res. 5, 2313-2319.ULLRICH, A., SHINE, J., CHIRGWIN, J., PICTET, R.,TISCHER, E., RUTTER, W. J., AND GOODMAN,H. M. (1977). Rat insulin genes: Construction ofplasmids containing the coding sequences. Science196, 1313- 1319.VALENZUELA,~., BELL,G. I.,VENEGAS,A., SEWELL,G. T., MASIARZ, F. R., DEGENNARO, L. J., WEIN-BERG, F., AND RUTTER, W. J. (1977). RibosomalRNA genes of Saccharomyces cerevisiae. II. Phys-ical map and nucleotide sequence of the 5Sribosomal RNA gene and adjacent intergenic re-gions. J. Biol. Chem. 252, 8126-8135.