Mutational analysis of the operators of bacteriophage lambda

Flashman, S.M.

doi:10.1007/bf00379730

Cited by 31 publications

(9 citation statements)

References 25 publications

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“…Repressor and Cro respond differently to operator mutations (refs. [14][15][16] and unpublished data), and our experiments indicate that the arm is responsible for this difference in at least three cases. Two of these operator mutations affect base pairs not contacted by the arm (6), yet the arm is partly responsible for their effect on repressor binding.…”

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confidence: 58%

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NH2-terminal arm of phage lambda repressor contributes energy and specificity to repressor binding and determines the effects of operator mutations.

Eliason¹,

Weiss²,

Ptashne³

1985

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

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Several lines of evidence indicate that the phage X repressor recognizes its operator by using, in part, an a helix (the "recognition helix"), which it inserts into the major groove of DNA. In addition to its recognition helix, X repressor has an "arm," consisting of the first six amino acids, that wraps around the DNA helix. We constructed plasmids that, in Escherichia coli, direct the expression of derivatives of A repressor that lack the NH2-terminal one, three, six, or seven amino acids. We studied these modified proteins in vivo and in vitro, and from our results we argue that the arm: (i) contributes a large portion of the binding energy; (ii) helps to determine sequence specificity of binding and, in particular, the relative affinities for two wild-type binding sites; (iii) determines entirely repressor's response to one operator mutation (a "back-side" mutation); (iv) magnifies repressor's response to other operator mutations ("front-side" mutations); and (v) increases the sensitivity of repressor binding to salt concentration and temperature.Models based upon x-ray crystallographic studies (1-3) and upon sequence homologies (4,5) suggest that many prokaryotic regulatory proteins recognize their binding sites by a common mechanism. According to these models (6-8), such proteins make sequence-specific contacts by inserting an a helix, the "recognition helix," into the major groove of DNA. In one case it has been shown that replacing the recognition helix from one protein with the recognition helix of another alters the binding specificity (9). Phage X repressor is unusual because, in addition to a recognition helix, it also bears a flexible NH2-terminal "arm" that consists of the first six amino acids and evidently wraps around the DNA (10). According to the current picture (6), each monomer of a repressor dimer inserts a recognition helix into the major groove on one face of the DNA helix, the "front side." The two arms of the dimer wrap around the DNA, making specific contacts in the major groove on the opposite face, the "back side," and also making nonspecific contacts to the DNA phosphates. Phage X Cro protein, which lacks an arm, binds to the same operator sites as does repressor. Cro also binds as a dimer, inserting a recognition helix from each monomer into the major groove on the front side of the DNA helix, but does not contact the back side of the operator (7).Previous experiments have shown that phage X repressor derivatives with defects in the arm have a reduced affinity for operator. A point mutation changing Lys-4 to Gln-4 greatly reduced repressor binding (11). Proteolytic removal of the NH2-terminal three amino acid residues decreased the affinity of a repressor fragment [the NH2-terminal domain (12)] for an operator site and altered the ability of the same repressor fragment to protect guanines in the operator from methylation by dimethyl sulfate (10). Since removal of the first three amino acids does not change the global conformation of the protein (13), alterations made to the...

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confidence: 58%

“…Plasmids carrying the initiation codon in the same reading frame as the cI gene conferred immunity to XcI7 and/or directed the production of a large amount of a 26,000-dalton protein. Restriction (14) were from this lab's strain collection.…”

Section: Methodsmentioning

confidence: 99%

NH2-terminal arm of phage lambda repressor contributes energy and specificity to repressor binding and determines the effects of operator mutations.

Eliason¹,

Weiss²,

Ptashne³

1985

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

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“…To confirm the predicted functions of these regions of the wEf11 genome, and to develop a potentially useful agent for phage therapy, we wished to determine whether deletion or replacement of these sites in phage wEf11 would result in a derivative virus with virulent rather than temperate properties. There is substantial precedent for temperate to virulent conversion of phage by modification of these genomic sites (Bailone & Devoret, 1978;Flashman, 1978;Donnelly-Wu et al, 1993;Bruttin & Brüssow, 1996;Ford et al, 1998).…”

Section: Discussionmentioning

confidence: 99%

Genetic modifications to temperate Enterococcus faecalis phage ϕEf11 that abolish the establishment of lysogeny and sensitivity to repressor, and increase host range and productivity of lytic infection

et al. 2013

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“…The classical vir mutants were selected as variants that form plaques on a lawn formed by a lysogen (23). Later studies demonstrated that two mutations in O R and one mutation in O L , or one mutation in O R and two mutations in O L , are necessary and sufficient for virulence (12).…”

Section: Isolation Of Virulent Mutantsmentioning

confidence: 99%

The Operator and Early Promoter Region of the Shiga Toxin Type 2-Encoding Bacteriophage 933W and Control of Toxin Expression

2004

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The genes encoding Shiga toxin (Stx), the major virulence factor of Shiga toxin-producing Escherichia coli, are carried in the genomes of bacteriophages that belong to the lambdoid family of phages. Previous studies demonstrated that induction of prophages encoding stx significantly enhances the production and/or release of Stx from the bacterium. Therefore, factors that regulate the switch between lysogeny and lytic growth, e.g., repressor, operator sites, and associated phage promoters, play important roles in regulating the production and/or release of Stx. We report the results of genetic and biochemical studies characterizing these elements of the Stx-encoding bacteriophage 933W. Like , 933W has three operator repeats in the right operator region (O R ), but unlike and all other studied lambdoid phages, which have three operator repeats in the left operator region (O L ), 933W only has two operator repeats in O L . As was observed with , the 933W O R and O L regions regulate transcription from the early P R and P L promoters, respectively. A lysogen carrying a 933W derivative encoding a noncleavable repressor fails to produce Stx, unlike a lysogen carrying a 933W derivative encoding a cleavable repressor. This finding provides direct evidence that measurable expression of the stx genes encoded by a 933W prophage requires induction of that prophage with the concomitant initiation of phage gene expression.Shiga toxin-producing Escherichia coli (STEC) strains are a group of food-borne pathogens that can infect humans, causing a range of illnesses including hemorrhagic colitis and lifethreatening sequelae such as hemolytic uremic syndrome (26, 50). Shiga toxins (Stx), a group of cytopathic toxins first identified in Shigella dysenteriae (reviewed in reference 39), are essential virulence factors of STEC, eliciting many of the pathological features associated with STEC infections (42). Stx holotoxins, classified as AB 5 toxins, are composed of one enzymatic A subunit and five receptor-binding B subunits (reviewed in reference 38). The A subunit is an rRNA N-glycosidase, catalyzing the removal of a specific adenine residue from the 28S rRNA of the 60S ribosomal subunit, resulting in inhibition of protein synthesis in the target cell (48). STEC produce two types of Stx proteins: Stx1, which is essentially identical to Stx of S. dysenteriae, and Stx2, which differs in sequence but not in function from Stx1 (38).In most identified STEC strains, the toxin genes, stxAB, are located in the genomes of prophages that resemble the coliphage (7,22,24,27,40,58). Lambdoid phages share similar genome organization and schemes of transcription control (3,20). The prophage remains in a quiescent state due to binding of the cI-encoded repressor protein to the right and left operator sites, in this way inhibiting transcription from the phage early promoters P R and P L , respectively (47). Prophage induction, which leads to lytic growth, results from removal of repression. This event occurs primarily through RecA-mediated represso...

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Mutational analysis of the operators of bacteriophage lambda

Cited by 31 publications

References 25 publications

NH2-terminal arm of phage lambda repressor contributes energy and specificity to repressor binding and determines the effects of operator mutations.

NH2-terminal arm of phage lambda repressor contributes energy and specificity to repressor binding and determines the effects of operator mutations.

Genetic modifications to temperate Enterococcus faecalis phage ϕEf11 that abolish the establishment of lysogeny and sensitivity to repressor, and increase host range and productivity of lytic infection

The Operator and Early Promoter Region of the Shiga Toxin Type 2-Encoding Bacteriophage 933W and Control of Toxin Expression

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