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Specifically Abolish Regulation by the Cytr Repressor In Proc. Nati. Acad. Sci. USA Vol. 88, pp. 4921-4925, June 1991 Biochemistry Single amino acid substitutions in the cAMP receptor protein specifically abolish regulation by the CytR repressor in Escherichia coli (protein-DNA interaction/protein-protein interaction/positive control/nucleoside and deoxynucleoside metabolism) LOTTE S0GAARD-ANDERSEN*, ALEXANDER S. MIRONOVt, HENRIK PEDERSEN*, VITALY V. SUKHODELETSt, AND POUL VALENTIN-HANSEN*: *Department of Molecular Biology, Odense University, Campusvej 55, DK-5230 Odense M, Denmark; and tInstitute of Genetics and Selection of Industrial Microorganisms, Moscow 113545, U.S.S.R. Communicated by Donald M. Crothers, February 8, 1991 (receivedfor review October 5, 1990) ABSTRACT Promoters in Escherichia coli that are nega- tively regulated by the CytR repressor are also activated by the -116 -93 -40 +1 +98 cAMP receptor protein (CRP) complexed to cAMP; as a characteristic, these promoters encode tandem binding sites for l l cAMP-CRP. In one such promoter, deoP2, CytR binds to the region between the tandem CRP binding sites with a relatively CRP-2 CRP-1 -10 deoC low affinity; in the presence of cAMP-CRP, the however, FIG. 1. Schematic map of the deoP2 promoter. Coordinates are repressor and activator bind cooperatively to the DNA. Here in base pairs; + 1 refers to the start site for transcription as indicated we have investigated this cooperativity by isolating mutants of by the arrow. The hatched bar labeled deoC indicates the 5' end of the CRP protein that abolish CytR regulation without exhib- the deoC gene. The remaining hatched bars indicate regions pro- iting a concomitant loss in their ability to activate transcription. tected by cAMP-CRP in DNase I protection experiments (CRP-1 and Four different, single amino acid substitutions in CRP give rise CRP-2) and the -10 region of the deoP2 promoter. The open bar to this phenotype. These amino acids lie in close proximity on indicates the binding site for the CytR repressor. the surface of the CRP tertiary structure in a portion of the protein that is not in contact with the DNA. In vitro analyses of in deoP2 in the absence of cAMP-CRP. In the presence of one of the CRP mutants show that it interacts with the DNA in cAMP-CRP, however, the affinity of CytR for this sequence a manner indistinguishable from wild-type CRP, whereas its is greatly increased (100-fold) and, moreover, the two pro- interaction with CytR is perturbed. These results strongly teins bind cooperatively to deoP2. Hence, CytR relies on indicate that cooperative DNA binding of CytR and cAMP- interactions with DNA-bound cAMP-CRP complexes in or- CRP is achieved through protein-protein interactions. der to bind strongly to deoP2, and cAMP-CRP may be viewed as an adaptor that allows CytR to associate with deoP2 (10). The cAMP receptor protein (CRP) complexed to cAMP binds In this report we have addressed the question of how cAMP- at specific sites in promoters to regulate initiation of tran- CRP and CytR interact at deoP2 by isolating CRP mutants scription of many operons in Escherichia coli. The complex that severely decrease CytR regulation. stimulates transcription in the majority of these systems, either as sole activator or as a coactivator. At other promot- MATERIALS AND METHODS ers cAMP-CRP acts negatively either directly by blocking the access of RNA polymerase or indirectly by stimulating the In Vitro DNA Manipulations. Isolation of plasmid DNA, synthesis of an antisense RNA (for review, see ref. 1). cloning, transformation of E. coli, gel analyses of recombi- The CytR repressor regulates initiation of transcription nant plasmids, and sequencing were performed as described from several operons whose products are involved in the by Maniatis et al. (11). uptake and catabolism of nucleosides and deoxynucleosides Bacterial Strains and Growth Media. All strains are E. coli in E. coli (2). All CytR-regulated promoters studied are K-12 derivatives: S0928 (Adeo, Alac, cytR+); S0929 (as S0928 activated by the cAMP-CRP complex, and all of those but cytR-) (12); S01316 [araD139, AlacU169, cod, rpsL, thi, analyzed in detail (deoP2, cdd, tsx, cytR) contain tandem cdd::-AMu-::Apl(209), cytR+]; S01319 (as S01316 but cytR-) binding sites for cAMP-CRP (3-6). The deoP2 promoter is (13); S01552 [Adeo, Alac, tsx::-AMu-::Apl(209), cytR+]; activated 30-fold by cAMP-CRP and repressed 10-fold by S01553 (as S01552 but cytR-). All ofthese strains were made CytR. In vivo analyses ofdeoP2 have shown that cAMP-CRP Acrp by P1 transduction using a P1 lysate grown on BRE2055 activation takes place from the CRP-1 target (Fig. 1). On the [A(argF-lacU169), A(crp)96 zhd-732: :Tn10] (14); RM1036 other hand, CytR regulation is dependent on CRP-1 and [A(lac-pro), thi, rpsL, supE, endA, sbcB, r-m-, mutD5, CRP-2 and on the length and the sequence of the region zaf-13::TnJO/F'traD36, proAB, lacIq, AlacM15] (isolated by between the two CRP sites. Furthermore, CytR only regu- R. Maurer and obtained through E. Bremer, University of lates initiations of transcription from deoP2 in the presence Konstanz) was used for mutagenesis as described (15). of cAMP-CRP (7-9). Plasmids. pJEL134 encodes deoP2 information spanning Consistent with these in vivo results, we have recently from position -116 to +98 (Fig. 1) (7). p122-002 encodes a shown in vitro that even though CytR exhibits an intrinsic 550-base-pair (bp) EcoRI-EcoRV fragment spanning the en- DNA binding capacity of its own, it only interacts weakly tire cytR promoter fused to lacZ in pJEL122 (7). p246-6 with a sequence located between the two CRP binding sites contains an EcoRI-Sma I fragment from pSKS106 (16) span- ning the entire lac promoter and cloned in the single copy The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: CRP, cAMP receptor protein; wt, wild-type. in accordance with 18 U.S.C. §1734 solely to indicate this fact. fTo whom reprint requests should be addressed. 4921 Downloaded by guest on October 1, 2021 4922 Biochemistry: S0gaard-Andersen et al. Proc. Natl. Acad. Sci. USA 88 (1991) Table 1. Nucleotide and amino acid substitutions in crp mutants Nucleotide Amino acid Number of sequence* substitution* isolates Position in secondary structuret CAT-221 CGT His-17 -- Arg 1 Last amino acid in a-helix A TGC-224 CGC Cys-18 -. Arg 4 First amino acid in B-sheet 1 GTA-494 - GCA Val-108 Ala 1 First amino acid in turn between a-helices B and C CCG-500 TTCG Pro-110 - Ser 2 Last amino acid in turn between a-helices B and C *Numbering of base pairs and amino acids is according to ref. 23. Substituted base is indicated by italic. tDenomination of secondary structures is according to ref. 24. number lacZ fusion vector pJEL246 (unpublished). The phenotype. Plasmid DNA from 20 independently isolated pML31 derivatives encoding the crp alleles were constructed Lac' colonies was retransformed to cytR+ strains encoding by cloning 3.5-kilobase (kb) BamHI fragments containing the lacZ fused to four different CytR-regulated promoters-i.e., entire crp between the two BamHI sites in pML31 (17). deoP2, cdd, tsx, and cytR-to test whether the mutations Gel-Retardation Experiments. See refs. 18 and 19 for de- conferring the CytR- phenotype were plasmid encoded. Of tails. Reaction mixtures contained 10 mM Tris HCl (pH 7.8), the 20 plasmids, 8 conferred a CytR- phenotype in all four 50 mM KCI, 0.5 mM EDTA, 50 ,ug of bovine serum albumin tester strains, whereas none of the remaining 12 plasmids per ml, 1.0 mM dithioerythreitol, 0.05% Nonidet P-40, 15 ,ug perturbed CytR regulation. A sequence analysis of the crp of competitor DNA per ml [supercoiled pGEM4 (obtained alleles from these 8 plasmids revealed four different muta- from Promega)], and 30 ng of a 32P end-labeled 308-bp tions (Table 1). fragment per ml encoding the entire deoP2. Purified CRP In Vivo Analysis ofCRP Mutants. To analyze the phenotype proteins, CytR extract, and cAMP were added as described of the CRP mutants in vivo at conditions that closely reflect in the text. Electrophoresis was performed in 5% acrylamide the wt situation, the four mutant crp alleles and crp-wt were gels using 10 mM Tris-HCI, pH 7.8/1 mM EDTA containing cloned in plasmid pML31, an F plasmid derivative exhibiting cAMP at the concentrations used in the reactions as electro- a copy number of one per genome equivalent. The CRP phoresis buffer. The fragment employed was isolated from mutants were tested for their competence to activate tran- p13-134 (9) as a 308-bp Xba I-Pvu II fragment; the centers of scription by introducing the five pML31 derivatives into CRP-1 and CRP-2 are positioned 149 bp and 202 bp from the cytR- strains encoding gene fusions between IacZ and Xba I end, respectively. deoP2, cdd, tsx, cytR, or the lac promoter. As crp on the DNase I Footprinting. DNase I footprinting experiments chromosome has been deleted in all tester strains, these were performed as described (20). strains only contain the CRP protein encoded by the pML31 Proteins. CRP proteins were purified as described (21). derivative. All of the CRP mutants activate as efficiently as CRP concentrations were determined spectrophotometri- CRP-wt; in fact, the CRP protein with the Cys-18 -* Arg cally employing E278 = 4.1 x 104 M/cm (22). CytR extract was substitution activates deoP2 and tsx more efficiently (Table prepared from a strain overproducing CytR by ammonium 2). sulfate precipitation (unpublished).
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