Bacteriophage λ protein cII binds promoters on the opposite face of the DNA helix from RNA polymerase

Ho, Yen-Sen; Wulff, Daniel L.; Rosenberg, Martin

doi:10.1038/304703a0

Cited by 117 publications

(75 citation statements)

References 26 publications

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“…In this regard, the action of RdgB probably is very similar to other activator proteins, such as lambda CI and cII proteins (Ho et al, 1983 ;Hochschild et al, 1983) as well as E. coli CAP, AraC, MalT and OmpR (Lee et al, 1981 ;Raibaud & Schwartz, 1984). The latter proteins are known to bind sequences within or upstream of the It is important to note that in Er.…”

Section: Structural Features Of the Pnla Promotermentioning

confidence: 99%

Activation of the Erwinia carotovora subsp. carotovora pectin lyase structural gene pnIA: a role for RdgB

et al. 1997

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The activation of pectin lyase (Pnl) production in Enwinia carotovora subsp. carotovora strain 71 occurs upon DNA damage via a unique regulatory circuit involving r e d , d g A and rdgB. In a similar Pnl-inducible system reconstituted in Escherichia coli, the rdgB product was found to activate the expression of pnlA, the structural gene for pectin lyase. The kinetic data presented here also show that transcription of pnlA followed that of rdgB in Er. carotovora subsp. carotovora, indicating a temporal order of gene expression. By deletion analysis w e have localized the promoter/regulatory region within a 66 bp DNA segment upstream of the pnlA transcriptional start site. This region contains the -10 consensus sequence but not the sequences corresponding to the Em coli -35 region. For DNA-binding studies, rdgB was overexpressed in E. coli and a 14 kDa polypeptide was identified as the gene product. RdgB from crude extracts or a purified preparation caused an identical gel mobility shift of a 164 bp DNA segment containing the pnlA promoter/regulatory region. Utilizing DNase I protection assay the RdgB-binding site was localized between nucleotides -29 and -56, i.e. overlapping the position of the putative -35 box. The findings reported here, taken along with our previous observation that the d g B product is required for pnlA expression, establishes that d g B encodes a transcriptional factor which specifically interacts with the pnlA promoter/regulatory region.

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Section: Structural Features Of the Pnla Promotermentioning

confidence: 99%

Activation of the Erwinia carotovora subsp. carotovora pectin lyase structural gene pnIA: a role for RdgB

et al. 1997

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“…DNA protection experiments and mutational analyses demonstrated that the cII binding site is comprised of tetranucleotide repeat sequence, TTGCN6TTGC (Ho et al 1983). In the presence of cII protein, RNA polymerase binds cooperatively to the promoter regions of PP, E, Pv and PaQ-The polymerase contacts occur primarily on the opposite face of the DNA helix from where cII binds and, thus, cII and RNA polymerase appear to sandwich the DNA between them [Ho et al 1983) The cII protein is different from several other wellcharacterized sequence specific DNA-binding proteins in that it recognizes a direct repeat sequence rather than a DNA sequence with twofold rotational symmetry, as is the case with the lambda repressor (cI) protein and Cro protein and the Escherichia coli CRP protein, Trp repressor, and Lac repressor [Gilbert and Maxam 1973;Dickson et al 1975, 19771 Dykes 1975Maniatis et al 1975;Gilbert et al 1976;Bennett et al 1977; Humayun et al 19771 Johnson et al 19781. At present, we do not know whether the cII tetramer has a twofold axis of symmetry or an asymmetric arrangement.…”

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confidence: 99%

“…The third promoter, PaQ, is thought to direct the synthesis of an antisense RNA, the function of which is to reduce the expression of the Q gene product (Ho and Rosenberg 1985;Hoopes and McClure 1985), necessary for the expression of all phage late genes during lytic growth. Because cII binding and transcription activation of P~, PI, and PaQ occur at essentially the same cII levels (Ho et al 1983;Ho and Rosenberg 1985), these three promoters function in concert to achieve the lysogenic growth response of the phage.…”

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Identification of the DNA binding domain of the phage lambda cII transcriptional activator and the direct correlation of cII protein stability with its oligomeric forms.

Ho¹,

Mahoney²,

Wulff³

et al. 1988

Genes Dev.

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The bacteriophage ?~ transcriptional activator protein cII is a DNA-binding protein that coordinately regulates transcription from phage promoters important for lysogenic growth. We have genetically and structurally characterized more than 80 different single amino acid substitutions in this 97-amino-acid protein. A subset of 25 of these variant proteins was utilized for detailed biochemical analysis, which allows us to define specific domains critical for sequence-selective DNA recognition, nonspecific DNA binding, and protein oligomerization. The mutation studies also demonstrated the remarkable correlation of oligomeric structure of cII protein to its stability within the bacterial host. An Escherichia coli HtpR-strain has been identified that greatly stabilizes these highly unstable cII mutants.

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“…Other regulatory proteins are also known that bind to DNA but do not recognize symmetric sequences. Among these are, for example, the X clI protein (39). Furthermore, the product of a gene involved in the regulation of lysogeny in the temperate B. subtilis phage 4105 was shown to interact with a 14-bp-long asymmetric operator site (P. Dhaese, personal communication).…”

Section: Discussionmentioning

confidence: 99%

Multiple repressor binding sites in the genome of bacteriophage P1.

Velleman¹,

Dreiseikelmann²,

Schuster³

1987

Proc. Natl. Acad. Sci. U.S.A.

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After digestion of bacteriophage P1 DNA with EcoRI in the presence of P1 repressor, 6 repressor binding sites were identified in 5 of 26 EcoRI fragments. Binding sites were localized by the decreased mobility of DNA fragment-repressor complexes during electrophoresis and by DNase protection ("footprinting") analysis. The repressor binding sites, or operators, comprise a 17-base-pair-long consensus sequence lacking symmetrical elements. Three operators can be related to known genes, whereas the function of the others is still unknown. The mutant P1 bac, rendering ban expression constitutive, is identified as an operator-constitutive mutation of the ban operon.P1 is a temperate bacteriophage with a genome size of about 90 kilobases (kb). In the prophage state the proviral DNA is maintained as a plasmid, and the vegetative P1 functions are repressed. Repression is accomplished by a phage-specific repressor, the product of the cl gene, which is located at the far right side of the P1 genetic map in EcoRI restriction fragment 7 (P1:7) (1, 2). Partially purified P1 repressor binds in vitro to at least two regions near cl within BamHI fragment 9 that itself is located within P1:7 (3, 4).The binding sites close to the cl gene, however, are not the only region at which the P1 repressor acts. The latter can also repress in vivo the expression of the P1 ban gene, which is located in P1:3 (5, 6). Furthermore, P1:14 also contains a promoter repressible by the product of cl (7). Together these results reveal that the P1 cl repression system must differ from that of other temperate phages such as A, P2, and P22, in which only promoters adjacent to the repressor gene are repressed (7).During our studies on the regulation of phage P1 ban expression we have localized by indirect methods a region 5' upstream of the ban gene within P1:3 at which the P1 repressor acts (8). Highly purified P1 repressor protein binds to this region in vitro (H.S., unpublished data). Now in a systematic search for other repressor binding sites in the phage genome, EcoRI-digested P1 DNA is incubated-with repressor, and binding regions are identified by the decreased mobility of EcoRI fragment-repressor complexes during electrophoresis (9,10). Thus binding regions were detected in P1:7 and P1:14, as expected and, in addition, in P1:9 and P1:11. Moreover, the results of DNA sequence and DNase protection analyses reveal an asymmetric 17-base-pair (bp)-long consensus sequence for the P1 repressor binding site. MATERIALS AND METHODSBacteria, Phage, and Plasmids. Escherichia coli K-12 strains used included the following: C600, HB101 (recA13) (11), NY58 (dnaBI07, recA56) (12), JM101 (13), N100 (galK, recA) (14), DW101 (sup'), and DW103 (supD) (15). For the cloning of P1 DNA fragments containing the ban operon (8) the recipient bacteria C600, HB101, NY58, and JM101 carried the plasmid pKT101-P1:7 harboring the P1 repressor gene (8). Strains DW101 and DW103 were transformed by plasmid pBR325-P1:11 for marker rescue tests (2).Phage used were M13mp8/9 (16)...

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Bacteriophage λ protein cII binds promoters on the opposite face of the DNA helix from RNA polymerase

Cited by 117 publications

References 26 publications

Activation of the Erwinia carotovora subsp. carotovora pectin lyase structural gene pnIA: a role for RdgB

Activation of the Erwinia carotovora subsp. carotovora pectin lyase structural gene pnIA: a role for RdgB

Identification of the DNA binding domain of the phage lambda cII transcriptional activator and the direct correlation of cII protein stability with its oligomeric forms.

Multiple repressor binding sites in the genome of bacteriophage P1.

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