Praele Acad. Sci. USA
`Vol. 79. pp. 40204024. July 1982
`Biochemistry
`
`Genes- encoding Escherichia coli aspartate transcarbarnoylase:
`The pyrB—pyrI operon-
`(nanra-iptional regulation/clonth and pyrl genes/map puttionot'gme mending resnlltnwdnins/intercistronic regtur)
`
`C. DAVID PAUZA', MICHAEL J. liners. MARC NAVRE, AND H. K. SCHACHMANl'
`Dem: oi'Molecular urology and Virus laboratory. Wendell u. Stanley run. unwary aroma. Berkeley. Calilbr-niam
`
`P
`(8). In addition.
`A hages capable of tr'arrsducing the
`chain (11), but the dlstancebetvlreen
`py
`r8 gene also encode ther
`pyrB and
`l is uncertain. Although the genes For the blosyn-
`thetic
`y (pyrB, ,1er. mm. pyrE, and pyrF) respond
`in a coordhrate manner to uracil starvathn (12), they map at
`difl'erent positions (13). The increase in ATCase activity, re-
`fleeting the elevated and balanced expression ol'pyrB and pyrl.
`is at least 10-fold greaterthan that ofthe other enzymes (12, 14).
`Support for the suggestion that new and
`are part ofthe
`
`same transcription unit stems hum two ingendent sets of
`
`the kinetics of
`experiments. Perbal and Herve (7) determin
`incorporation ofradioactively labeled amino acids into the e and
`rchains during and immediately after uracil starvation. and on
`the basis of the results they proposed that synthesisol'both
`types of polypeptide chains was directed by a polycistronic
`musenger RNA in 8. call. Further evidence for an operon
`structure was provided by Syvanen and Roth (8) in their dem-
`onstration of the polarity of pyrB chain-terminating mutations
`on the synthesis of r chains in S. typhimurium.
`In order to eliminate uncertainty regardtn the organintion
`ol' the pyrli and pyrl genes and the control
`their expression,
`we have analyzed the elI'ect oi pyrB deletion mutations on the
`synthesisoltherpolypeptidechains. Thesestudies havesbown
`that pyrl 3 subject to the same transcriptional regulation as
`fiayrfl even in the absence ofthe pyrB product. In addition, we
`ve determined the position of the pyrl gene by DNA se-
`quence analysis of the intercistroaic region between pyrB and
`pyrl: These results establish that the contiguous p r8 and pyrl
`genesconstituteasingletransu-i
`' naluniten
`in thecand
`rchainsolE.coliATCase. W
`s
`
`r
`
`MATERIALS AND METHODS
`
`Bacterial Strains and Their Construction. All S. typhirnu-
`riurn strains listed in Table l are derivatives of L1! except
`'I'RBSOO. which was derived fiom [.17. The pyrH'lm mutation
`results in a
`ly defective UMP ltinase; as a consequence
`the levels UDP and UTP are reduced. resulting in yeatly
`increased production of-A'l‘Case (9. 15-17). Mutation 1:9er
`is a deletion that removes all of pyrfl (8). The F393-episome is
`derived horn FIE (F' mloc)ol£. colt K—12and contains the
`E. coli pyril (17). M '
`transductional methods. and conjo-
`grtional transfers were described earlier(17). High-titer p
`d
`P22 lysates for mapping eslpcriments were concentrated lO-l'o
`to yield titers ofabout- IllI plaque-fanning units]ml; they were
`stored in T-2 butler (18).
`
`Abbreviations: A'ICase. aspartate nursearbamoylase; e, anlglc
`peptidechain; r, regulatorypolypeptidechain: C. catslyticru unit. R,
`regulatory subunit; Pyrfl Midine-independent: 1dr. ldlobase.
`' Present address: Med
`Council Laboratory of Molecular
`
`Contributed by Howard 1:. 5mm... April 9. 1982
`
`In both Escherichia coli and Salmonella typhi-
`ABSTRACT
`murhnn there is approximately balanced synthesis of the six cat-
`alytieand sis regulatory polypeptide chains oithe regulatory enw
`zyma aspartate transcarba
`(earbamoylphosphatesbu-
`transl'erase, £011.12). 111k control is achieved
`-pyrl genes, which encode the catalytic
`'
`and regulatory chains, respectively. Evidence for this single tran-
`su'iptional unit was obtained by a studyol'various deletion mu-
`ahmmdhomalreDNAuqmdgfltlaehmm
`tween
`an
`1. One port!
`a, pyrB?
`,
`a normal
`alligator? chains even thoughit removed a sub-
`stantial portion of the pyrB gene. Another deletion. pyrBfltl.
`sharesasimilarterminus atoneendwlthinpyrli,butthepromotet'
`regionis removedrln-thisdeletionmutation, thereisnoprodue
`lion of the regulatory polypeptide, indicating that a single region
`adjacent to pyrB'contmls transcription ol'pprl as well. Molecular
`cloningand subsequent DNA analysis demonstrated that the pol-B
`andpyrlgenesarecontiguouswithpyrlasthedistal geneinthe
`w. The cistnons are separated hybr: 1°55nudeotide um
`region containing a sequence caps e
`interacting
`165 ribosomal RNA and allowing translation oi the pyrt else-on.
`
`It has been known for many years that the allosteric enzyme
`asparlate transurhamoylase (A‘l‘Case; aspartate carbamoyl-
`hansferase; carbamoylphosphateztraspartate msbamoyltrans-
`i'erase; EC 2.1.8.2) from Escherichia coli is an oligomer com-
`posed olsis catalytic (c) and six regulatory (r) polypeptide chains
`(1-6). Moreover. there is approximately balanced biosynthesis
`olthe chains in both E. coli (7) and Salmonella typhimuriunr»
`(8). These observations led in the suggestion that the structural
`genes for the cand rchains (pyrB and pyrl. respectively) are
`as an operon (8) and that biosynthesis involves a
`pot:chmessengerRNA(7). However. precise lmowledge
`location ofthe purl geneand definitive evidence thatboth
`argues are to the same transu'iptional control have not
`avai
`e.
`A'l'Case of both E. coll and S. typhimurimn is composed of
`two tr'lmeric catalytic (C) subunits and three dimeric regulatory
`(R).subunits (1-5. 9. 10). isolated C trimers ofmolecular weight
`100,000 are catalytically active but insensitive to-the feedback
`inhibitor. CIT. and the activator. ATP (1), whereas the free R
`dimers of molecular weight 34,000 are devoid of catalytic ac-
`tivity but still bind both Cl? and ATP with high affinity (I).
`When C and. R subunits are mixed. reconstituted ATCase oi
`molecuhcwei
`t300.000 is fimned in high yield, and the com-
`plex exhibits
`allosteric properties of native ATCase (1).
`Several lines of evidence indicate that the owl gene is in
`close proximity to the»
`r]! gene. Single deletions in S. typin-
`nurrium eliminatep
`notion of bothc andr polypeptide chains
`
`
`
`charge
`blimflonoostsoithisarticleweredefi-ayedinpanby
`maLThbanhlemustflrerelorebehemhymukedmd‘doerm
`
`

`

`Table 1 Bacterial strains
`
`Genotype
`
`Strain
`S. Whistler-ion:
`HSMD err-312002 [0140! M798 prnAflfl pryer pyrH‘iOO
`H5223! dream fol-I01 #217798 pmAB47pyr8655pyrH700/
`F393 ioc‘ pro’ orgnw (P22 pyrB')
`H52255 car-512002 fold!!! hub?” pmABl? pyer pyrHi’OO/
`P893 ioc‘ pro+ rugF282fl‘niO (P22 pyrBMO)
`1132256 “12002 fol-101 lequ pruAB47 3123655 pyrH700/
`P393 lot" pro‘ orgHSZs’I‘nID (P22 pyrB‘)
`HS2273 rat-32002 foiJOI 1:11.0798 proABfl pyr3655 pyrH700/
`F393 lae‘ pro‘ orgH82:.’I‘n10 (1’22 pan-£743)
`rat-312002 fol-101 lequ prnAB47 pyrBBEfi pyrH700/
`F393 loc‘ pro‘ 922 pyrBi'H)
`H52308 fol-101 lequ preAB47 pyrBB55 pyrli700/
`F393 lar‘ pro‘ warms (P22 part-B748)
`H8230? fol-101 leuDi'98 praABfl pyr3655/
`F398 loc' pro+ orgflafltfl‘aifl (1’22 pyrB74dl
`TR32M‘ amtAI M47 pyer tip-130
`
`1182278
`
`Pop 1] fragment and both strands of the Msp 1 fragment were
`
`found to be present in the extracts in preliminary experiments.
`This procedure was followed in order to obtain the maximal
`sensitivity consistent with a broad range of detection. The re-
`constitution oiA'l‘Case was achieved by incubating the mixtures
`of subunits for 30 min at 30°C. In all experiments the labeled
`C subunit was in excess of any unlabeled C subunit in the ex-
`tracts, and not more than 50% of the “st-labeled subunit was
`converted to radioactively labeled ATCase. Unlabeled ATCase
`and C subunit were added as markers and the samples were
`subjected to electrophoresis in 5-cm polyacrylamide gels (5%).
`Alter electrophoresis the gels were stained and sliced. and the
`distribution of radioactivity in ATCase and C subunit was
`determined.
`Plasmid Construction. Restriction endonucleases were ob-
`tained from New England Biolabs and used according to the
`supplier's specifications. The bacterial strain ADllm5. mnying
`the A specialized hansducing phage ylrltim5 (Ad pyrB orgl ualS)
`as a prophage (20) was kind] provided by Alci Kilnichi. Bae
`age DNA was isolated y the procedure ofWu et a1. (21).
`Plasmid DNA was isolated by the allotline-NaDodSO. extrac-
`tion method of Bimboim and Duly (22). and the DNA was pu-
`rified further by centrihsgation in a CsCl/ethidium bromide
`gradient
`A molecular recombinant plasmid containing the pyrB gene
`was constructed by digestion of ylcl4m5 and 1333322 (24) DNA
`with the restriction endonucleases Psi l and Ecofll and sub-
`sequent ligation of the fragments with phage T4 DNA ligase.
`Plasmids were used to transform strain AT2535 (carrying the
`:1er allele) made competent by treatment with CaCI, (25).
`The resulting transfer-moms were selected as Pyr“ tetracycline-
`resistant colonies. One isolate was selected and the 7.4olcilobase
`(kb) plasmid, pDP7, was purified and analyzed by restriction
`enldonuclease digestion and electrophomis in 0.7% agarose
`ge s.
`The size of the DNA fragment containing the mud! gene was
`reduced by digesting plasmid pDP? with Ecolil and treating
`the linear DNA with .8013] double-stranded exonuclease (26)
`ibr 30 min at 30'C. incubation of the resulting DNA overnight
`with T4 DNA ligase yielded plasmid pDPB, which was used to
`transform AT25'35 cellsI selecting for pyrimidine prototrophy.
`Isolates were screened to ensure that they were tetracycline
`sensitive beause exonucleolytic digestion of pDP7 from the
`Ecolil site should result in the loss of tetracycline resistance
`(27). The ability of plasmid pDPB to encode both c and r poly-
`peptides was tested as follows: A derivative ol'TB3200 (carrying
`the rapt-8655 deletion) containing pDP8 was constructed. Cel-
`lular extracts were prepared and subjected to electrophoresis
`in 5% polyacrylamide gels. The mobilities of the enzymimlly
`active proteins were determined by using the actiVity stain de-
`scribed hy Bothwell (28). As little as 1 ng ofactive protein (ATC-
`ase or C subunit) can be detected readily by this procedure.
`Sequence Determination. A DNA fragment approximately
`650 base pairs in length was derived by digestion oipDPB with
`the restriction endonuclease Map 1 (Fig. 1) and was labeled at
`the 5' termini with ['y-nPlATP (Amersham. >5,“ Cl/mmol)
`and polynuclcotide itinase (P-L Biochemicals). The DNA
`strands were separated according to the procedure of Masam
`and Gilbert (29). In other experiments the plasmid was digested
`with Bgl I], and the 3' ends were labeled with the large frag-
`ment of DNA polymerase (Boehringer Mannheim) and m
`a"P—labeled deoxyribonucleoside triphosphates (Amersham.
`>8,000 Climmol) followed by digestion with Per: 1]. The re-
`sulting 050-base-pair fragment was isolated by polyacrylamide
`gel electrophoresis (20). Nucleotide sequences of the Hg! 11/
`
`Ar
`
`E coli
`£12535i pyrB59. nigHZ, thi-I . his-1, purFi , mil-2, ayl-T. molAi.
`urn-13, ice?! or tom, rye-18, 9 or 14. mm or ion-
`14, tar-28 or tax-25. A', A'. strle
`
`‘Kindlypravidedbyd. [LRotlt
`‘Dbtainart from theE. coli‘Genetic Stock Center (New Haven. LT).
`
`Isolation and Mapping of Deletion Mutants. Deletion mu-
`tants 1152255 (pyrB‘MO). 1152.273 (pyrB'MS), and 1152273
`3754) are spontaneous derivatives of HS2256 obtained by
`se acting for pyrB mutants as suppressors of arginine auxotro-
`phy (17). These deletion mutants are completely auxotropic for
`pyrimidines. Mapping was performed by transductional crosses
`using high-titer P22 stocks
`on point mutants and select-
`ing for the ability of the deletion mutants to form pyrimidine-
`independent (Pyr*) tnnsductnnts in spot tests on selective me-
`dia. Endpoints were confirmed by crosses using 100-200 pl of
`high-titer P22 steels and 100 pl olrecipient cells that had been
`concentrated 10-fold alter overnight growth in nutrient broth
`supplemented with a 2% vol of E medium (at 50 times the nor-
`mal concentration). The crosses were scored alter48 hr at 37°C.
`in crosses in which recombinatlon was possible at least 100 col-
`onies resulted, whereas negative results were indicated by a
`complete absence of Pyr“ transductants.
`Preparation of Cell Extracts. Bacteria for assays of R subunit
`were grown to late logarithmic phase in 25-ml cultures of E
`medium supplemented with 0.25% glucose. uracil at 20 pg]
`ml, and required amino acids at 100 Mm]. Extracts were pre
`pared as described by Syvanen and Roth (8). Protein concen-
`trations in the extracts were 0.8-1.5 mg/ml.
`Preparation of Radioactiver Labeled C Subunit. “514.c-
`beled C subunit was prepared by the method of Syvanen at at
`(19), using 0.5 mCt (1 Ci = 3.7 X 10’” becquerels) of Nam!
`Amarsham) and 40 pg of purified C subunit from E. coli ATC-
`ase. The specific activity of the labeled protein was 4.2 x 10‘
`m pg.
`Assay for it Subunit in Cell Extracts. The amount of 0 sub-
`unit in the extracts was measured by the procedure (assay 11)
`of Syvanen and Roth (B). Cell extracts (50 111) were treated with
`0.25 pmol oineohydrin (K a K) to dissociate an ATCase into
`free it and G subunits (1). After 2 min the bisu strate analog
`N-(phosphonacetyl)L-aspartate was added to yield a concen-
`tration of 20 Ml, followed by the addition of 1.5 pmol of 2.
`mercaptoethanol and '“l-labeled C subunit. The amount of
`I"’i~labeled C subunit was varied between 2 and B pg (1.5 x
`105 cpm per tube). depending upon the amount of R subunit
`
`
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