Lac repressor genetic map in real space

Pace, Helen C.; Kercher, M.A.; Lu, Ponzy; Markiewicz, Peter; Miller, Jeffrey H; Chang, Geoffrey; Lewis, Mitchell

doi:10.1016/s0968-0004(97)01104-3

Cited by 61 publications

(43 citation statements)

References 18 publications

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“…Together, the protection conferred by the catalytically inactive protein in sparing the chromosomal MraY and the existence of three classes of E-resistant MraY mutants may indicate that the fX174 R alleles of mraY encode proteins with different affinities for E. In this view, the F288L mutant is resistant because it binds E poorly, whereas the G186S and V291M mutants bind E with an affinity that is not distinguishable, at least in our assay, from that of the wt protein. These classes resemble the different classes of inducer insensitivity that have been observed in the Lac repressor, in which some mutations block inducer binding but others interfere with the inducer-mediated conformational change (Pace et al 1997). It will be interesting to exploit this system to select E mutants that overcome the mraY mutations, with the aim of using allele-specific suppression to map out point-to-point interactions between E and MraY.…”

Section: Ecmentioning

confidence: 80%

Genetic Analysis of MraY Inhibition by the ϕX174 Protein E

et al. 2008

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Protein E, the lysis protein of bacteriophage fX174, is a specific inhibitor of MraY, the phosphoMurNAc-pentapeptide translocase that catalyzes the synthesis of lipid I in the conserved pathway for peptidoglycan biosynthesis. The original evidence for this inhibition was the isolation of two spontaneous E-resistance mraY mutants. Here we report further genetic studies aimed at dissecting the interaction between E and MraY, using a genetic strategy that is facile, rapid, and does not depend on the availability of purified E, purified MraY, or its substrates. This system relies on the ability of mraY or its enzymatically inactive D267N allele to protect cells from lysis after induction of a chimeric lTE prophage. Using this approach, the MraY protein from Bacillus subtilis, which shares 43% sequence identity with the Escherichia coli enzyme, was found to interact weakly, if at all, with E. A potential E binding site defined by transmembrane domains 5 and 9 has been identified by isolating more mraY mutants resistant to E inhibition. Genetic analysis indicates that these E-resistant alleles fall into three classes on the basis of the affinity of the encoded proteins for MraY.

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Section: Ecmentioning

confidence: 80%

Genetic Analysis of MraY Inhibition by the ϕX174 Protein E

et al. 2008

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“…The three native cysteine residues in LacI (at positions 107, 140, and 281) were replaced with alanine; E36C or Q231C were each introduced by site-specific mutagenesis (Quickchange, Stratagene) of the Cys-less LacI gene. Previous data demonstrate that these mutants exhibit no phenotypic effect on LacI function (25). Wild-type LacI and protein mutants were expressed and purified, as described previously (28), except that the purification of mutant proteins was carried out in the presence of 2 mM DTT.…”

Section: Methodsmentioning

confidence: 99%

Tetramer opening in LacI-mediated DNA looping

Rutkauskas

Zhan²,

Matthews

et al. 2009

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

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Lactose repressor protein (LacI) controls transcription of the genes involved in lactose metabolism in bacteria. Essential to optimal LacI-mediated regulation is its ability to bind simultaneously to two operators, forming a loop on the intervening DNA. Recently, several lines of evidence (both theoretical and experimental) have suggested various possible loop structures associated with different DNA binding topologies and LacI tetramer structural conformations (adopted by flexing about the C-terminal tetramerization domain). We address, specifically, the role of protein opening in loop formation by employing the single-molecule tethered particle motion method on LacI protein mutants chemically cross-linked at different positions along the cleft between the two dimers. Measurements on the wild-type and uncross-linked LacI mutants led to the observation of two distinct levels of short tether length, associated with two different DNA looping structures. Restricting conformational flexibility of the protein by chemical cross-linking induces pronounced effects. Crosslinking the dimers at the level of the N-terminal DNA binding head (E36C) completely suppresses looping, whereas cross-linking near the C-terminal tetramerization domain (Q231C) results in changes of looping geometry detected by the measured tether length distributions. These observations lead to the conclusion that tetramer opening plays a definite role in at least a subset of LacI/DNA loop conformations. (1). This effect is due to the fact that LacI is a homotetrameric protein assembled as a dimer of dimers, with each dimer presenting a DNA-binding domain, enabling the tetramer to bind simultaneously to two operators (2) and form a loop in the intervening DNA ( Fig. 1 A and B). Consequently, LacI binding to one of the auxiliary operators increases the proximity of the remaining free DNA-binding domain to the primary operator, thus enhancing the probability of transcription blocking. Moreover, upon dissociation from the primary operator, the repressor is likely to remain bound to one of the auxiliary operators, avoiding diffusion into the cytosol and retaining proximity to the primary operator to facilitate rebinding. Thus, LacI-mediated DNA looping is fundamental for the efficiency of repression, as demonstrated by the drastic effects of deletion of auxiliary operators (3).In general, DNA looping is a recurring motif among several other DNA-binding proteins with different functions (4). The looping probability is quantified by the J m factor (5), defined as the local concentration of the LacI bound to one of the operators with respect to the remaining free operator. This factor is governed by the DNA-protein mechanics, as manifest in the profound dependence of in vivo repression (6) or in vitro cyclization efficiency (7) on interoperator spacing. Modulation of this property with the period of helical repeat is due to a large DNA-twisting-associated energetic penalty and, therefore, certain phase requirements for joining the loose DNA ends for cycliz...

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“…Using a ␤-galactosidase colorimetric assay, each protein with a single amino acid substitution had been tested for its ability to (1) repress transcription at the lac operator and (2) cease repression upon binding of IPTG, the inducer sugar. More than 50% of the sites were generally tolerant to substitutions, and the regions that were sensitive to amino acid replacements were primarily at the DNA and inducer binding sites and at the dimer interface (Pace et al 1997). We compared predictions from SIFT and the substitution scoring matrices with the resulting phenotypes from the substitutions examined in the mutagenesis studies.…”

Section: Comparison Of Sift With Blosum62 Predictions On Laci Mutatiomentioning

confidence: 99%

“…Amino acid side chains that have been identified as involved in interactions (Chuprina et al 1993;Bell and Lewis 2000) are labeled as follows: (double helix) those that interact with DNA, (double cylinders) those participating in the dimer interface. (Hexagons) Positions having six or more substitutions that are unable to respond to the inducer (Markiewicz et al 1994;Pace et al 1997). Many of the intolerant positions that were predicted to tolerate substitutions correspond to these query-specific positions.…”

Section: Comparison Of Sift With Blosum62 Predictions On Hiv-1 Proteamentioning

confidence: 99%

“…Prediction for neutral substitutions decreases only slightly, which indicates that the remaining protease sequences are diverse enough for prediction. The effect of 4004 substitutions was assayed for LacI (Markiewicz et al 1994;Pace et al 1997), 336 substitutions for HIV-1 protease (Loeb et al 1989), and 2015 substitutions for bacteriophage T4 lysozyme (Rennell et al 1991). These three data sets are used to test prediction performance.…”

Section: Comparison Of Sift With Blosum62 Predictions On Hiv-1 Proteamentioning

confidence: 99%

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Predicting Deleterious Amino Acid Substitutions

Ng¹,

Henikoff²

2001

Genome Res.

2,332

2,014

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Many missense substitutions are identified in single nucleotide polymorphism (SNP) data and large-scale random mutagenesis projects. Each amino acid substitution potentially affects protein function. We have constructed a tool that uses sequence homology to predict whether a substitution affects protein function. SIFT, which sorts intolerant from tolerant substitutions, classifies substitutions as tolerated or deleterious. A higher proportion of substitutions predicted to be deleterious by SIFT gives an affected phenotype than substitutions predicted to be deleterious by substitution scoring matrices in three test cases. Using SIFT before mutagenesis studies could reduce the number of functional assays required and yield a higher proportion of affected phenotypes. SIFT may be used to identify plausible disease candidates among the SNPs that cause missense substitutions.

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Lac repressor genetic map in real space

Cited by 61 publications

References 18 publications

Genetic Analysis of MraY Inhibition by the ϕX174 Protein E

Genetic Analysis of MraY Inhibition by the ϕX174 Protein E

Tetramer opening in LacI-mediated DNA looping

Predicting Deleterious Amino Acid Substitutions

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