`
`IPR2016-00710
`
`The Organization and Regulation of the pyrBl Operon
`in E. coil Includes a Rho- Independent Attenuator Sequence
`William D. Roof', Karen F. Foltermannt, and James R. Wild "2
`' Department of Biochemistry and Biophysics, and 2 Faculty of Genetics, Texas A &M University, College Station, Texas 77843 USA
`
`Summary. 1. The two polypeptide chains that comprise as-
`partate carbamoyltransferase
`in Eschen chia coli are
`encoded by adjacent cistrons expressed in the order, pro-
`moter- leader -catalytic cistron -regulatory cistron (p- leader-
`pyrBl). These two cistrons and their single control region
`have been cloned as a 2,800 base pair (bp) fragment (The
`minimal coding requirement for the catalytic and regulatory
`polypeptides is about 1,350 bp plus control regions). The
`genes contained by this fragment are subject to normal re-
`pression controls and thus possess the intact control
`regions.
`2. By deleting an internal fragment with specific restric-
`tion endonucleases, it was possible to construct shortened
`fragments which no longer produced the regulatory poly-
`peptide. In these cases the expression of the catalytic cistron
`was normal and subject to repression upon growth in the
`presence of uracil. Since the pyrB cistron retained transcrip-
`tional control, the regulatory polypeptide was not required
`for expression or control of the catalytic cistron. As ex-
`pected, the catalytic trima (M,= 100,000 dallons) from
`these deletion mutants had no effector response nor did
`it exhibit homotropic kinetics for aspartate. The enzyme
`was identical to the c3 trimer purified from the native ho-
`loenzyme by neohydrin dissociation.
`3. Insertion of Mu dl(lac Ap') into the structural region
`of pyrB had a negative effect on the expression of pyr!.
`This supports the idea that the catalytic and regulatory
`polypeptide chains of aspartate carbamoyl -transferase are
`encoded by a single bicistronic operon. Detailed restriction
`analysis of the cloned pyrBl region has produced a genetic
`map of restriction sites which is colinear with the published
`amino acid sequences of the two polypeptides. These maps
`indicate that the 3'- terminus of the catalytic cistron is adja-
`cent to the 5'- terminus of the regulatory cistron and sepa-
`rated by 10-20 bp.
`4. DNA sequence analysis of the 5'- proximal regions
`of pyrB! revealed that an extensive leader sequence sepa-
`rated the promoter and first structural gene pyrB. This lead-
`er of approximately 150 bp contains an attenuator sequence
`and the translational signals required for the production
`of a leader polypeptide of 43 amino acids.
`In this paper we describe the structural organization
`of pyrB!, and provide a detailed analysis of its regulatory
`region including its DNA sequence.
`
`Offprint requests and all correspondence to: J.R. Wild
`
`Introduction
`
`De novo pyrimidine (UMP) biosynthesis (see Fig. 1) in both
`Escherichia coli and Salmonella typhimuritan is regulated
`allosterically at aspartate carbamoyltransferase (ATCase;
`Gerhart and Pardee 1962) and carbamoylphosphate synthé-
`tase (CPSase; Pierard et al. 1965; Anderson and Meister
`1965) or transcriptionally throughout (carAB to pyrF) in
`response to fluctuations in endogenous nucleotide pools
`(Williams and O'Donovan 1973; Kelln et al. 1975;
`Schwartz and Neuhard 1975). While the evidence for the
`involvment of various nucleotide pools in the repression/
`derepression of the pyr genes is clear, extensive searches
`for a suitable aporepressor have not been successful
`(O'Donovan and Neuhard 1970; Kelln and O'Donovan
`1976). O'Donovan and Gerhart (1972) reported a putative
`pyrR which resulted in derepression of the pyr genes, but
`upon subsequent analysis it was found to be a mutation
`in UMP kinase (pyres) (Justesen and Neuhard 1975). This
`leaky mutant produced greatly reduced levels of UDP and
`UTP, thereby causing derepression of the de novo pathway.
`Kelln has isolated cis -acting mutations ("operator- like ")
`which are derepressed for pyrB (RA. Kelln, personal com-
`munication) and recently, Jensen et al. (1982) reported the
`derepression of pyrB in a strain of S. typhimurium possess-
`ing an altered RNA polymerase (mapping in the rpoBC
`gene cluster). While this observation suggests a regulator)
`role for RNA polymerase in the expression of pyrB, th
`regulatory control of pyrBf remains undefined. The preser
`study details the structural organization of the pyrBf region
`of the E. coli chromosome and presents evidence consistent
`with sequential expression of the catalytic and regulatory'
`polypeptides from a bicistronic operon (po pyrB, pyr!). Sim-
`ilar results describing a bicistronic operon encoding
`ATCase have been obtained for S. typhimurium (G. Mi-
`chaels and RA. Kelln submitted to Mol. Gen. Genet).
`The aspartate carbamoyltransferase (EC 2.1.3.2) of E.
`cols is a multimeric enzyme possessing allosteric control sites
`on regulatory polypeptides which are distinct from the cata-
`lytic subunits (Gerhart and Schachman, 1965). The ATCase
`of E. coli and other enteric bacteria (Wild et al. 1980) and
`yeast phosphofructokinase (EC 2.2.1.11; Laurent et al.
`1978) are the only reported multimeric enzymes comprised
`of catalytic subunits and separable regulatory subunits. The
`native holoenzyme of E. coli is a dodecamer composed of
`six identical catalytic polypeptides (functional as a trimer,
`c3) and six identical regulatory polypeptides (functional as
`
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`
`CATALYTIC POLYPEPTIDE
`REGULATORY POLYPEPTIDE
`Fig. 4. The colinear map of the pyrB -pyr! region and its polypeptides. Since class 11 restriction endonucleases recognize specific inverted
`palindromic sequences, it is possible to determine the corresponding di- and tri -peptides encoded by such DNA segments. For example:
`The Hpa I restriction site, 5'- GTTAAC -3', has the potential to encode N- Val -Mn when transcribed and translated in the first position
`reading frame; N- Leu -Thr in the second reading frame or a chain- terminating UAA in the third. Only one Val -Mn sequence is observed
`for the amino acid sequence of the regulatory polypeptide at residues 83/84 and the Leu -Thr combination is not observed. Similar
`logic applies for the Bgl 11 restriction sites, 5'- AGATCT 3', which unequivocally designates Asp -Leu at amino acid residues 57/58.
`Theoretically, the restriction of the DNA with Bgl Il and Hpa I should produce a fragmeit of 80 bp in length. This is presented
`in the top schematic figure. Thus the locations of the regulatory and catalytic cistrons are established accurately. It was possible
`to locate the unique position for all restriction endonucleases recognizing 6 or more base -pairs. Using endonucleases that recognize
`5 bp sites it was possible to estimate the location relative to DNA fragment sizes. By comparing the known polypeptide map to the
`restriction endonuclease map it was possible to determine that between 10-20 base pairs separate the catalytic and regulatory cistrons
`
`lytic trimers and regulatory dimers occurs only in the cyto-
`plasm and not on transcriptionally active polysomcs. Fur-
`thermore, the same study showed that there is less than
`five percent production of r in the absence of c. The results
`of our study support the earlier suggestion by Perval and
`Herve (1972) that the pyrB and pyrl cistrons comprise a
`bicistronic operon in which the catalytic cistron is promot-
`er- proximal.
`
`The Regulatory Polypeptide has no Role
`in the Expression of the Catalytic Cistron
`The plasmid pPB -h104 was subjected to internal deletion
`by Hpa! which removed approximately 2,500 bp including
`the carboxy- terminal region of pyrl. In a separate deletion,
`650 bp including the last two -thirds (95 amino acid resi-
`dues) of pyrl were removed. In each case, pyrB was respon-
`sive to typical repression conditions in the presence/absence
`of exogenous uracil (50 pg/ml) for both plasmids (Table 2).
`Relevant plasmids (Table 1) were characterized by acrylam-
`ide gel electrophoresis and by auxotrophic- characteristics.
`After the internal fragments were removed, the resulting
`plasmids were transformed into competent TB2 cells. The
`molecular weights of ATCase from the various plasmids
`were determined by chromatography on Sephadex G -200
`as described in earlier reports (Wild et al. 1980). The strains
`containing the catalytic pyrB and partial deletion of the
`pyrl cistron produced only catalytic polypeptides and all
`enzymatic activity was recovered as catalytic trimers (Me=
`100,000 dallons. Thus, the presence of a functional pyrl
`gene product is not required for normal pyrB expression
`and regulation.
`
`Analysis of the Regulatory Region of pyrBl
`by DNA Sequence Determination
`The DNA sequence of the promoter region of pyrB! is
`presented in Fig. 5. The nucleotide sequence contains three
`
`Table 2. Repression of ATCase formation in various plasmid con-
`structs of E. colt The specific activities are expressed as micromoles
`of carbamoylaspartate produced per minute reaction time per milli-
`gram of protein from a cell -free extract. The strains and plasmids
`are described in the text and Table 1. Repression index is calculated
`as the ratio of specific activity without uracil /specific activity with
`uracil for each strain
`
`Strain
`
`Genotype
`
`Specific
`activity
`
`Repression
`index
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`K12 (min)
`K12 (+U)
`TB2 (min)
`TB2:pPBh104 (min)
`TB2:pPBh104 (+ü)
`TB2:pPBc201 (min)
`TB2:pPUc201 (+U)
`TB2:pPBc202 (min)
`TB2:pPBc202 (+U)
`
`pyrs', pyr!'
`pyre ,pyrl'
`pyrB-, pyr!-
`pyrs', pyrr
`pyrB', pyrl*
`pyrB `, pyr!
`pyrs', pyrr
`pyrB',pyr!'
`pyrB`, pyrP
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`134
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`151
`39
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`1.7
`
`0
`3.8
`
`sites that are organized spatially along the DNA in agree-
`ment with the consensus "idealized" promoter sequence
`(Pribnow 1975; Gilbert 1976; Rosenberg and Court 1979).
`A classic RNA polymerase recognition site (Re) is centered
`ten base pairs ( -10) from the presumptive transcriptional
`initiation site designated I (bp= +1). A sigma "recognition
`site" (Ra) is located approximately thirty -five base pairs
`( -35) preceding the transcriptional initiation site. The Re
`sequence, TATAATG, represents the "idealized" Pribnow
`box which serves as the base specific contact sequence for
`the RNA polymerase core (Pribnow 1979). Twelve base
`pairs separate the Re sequence from Re, thus defining an
`ideal promoter sequence covering approximately 40 bp.
`This precedes the translation initiation of the catalytic poly-
`peptide of ATCase ( +153) which can be identified by fitting
`
`Merck Ex. 1075, Pg. 5
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