Construction of A Dimeric Repressor: Dissection of Subunit Interfaces in Lac Repressor

Chen, Jiawen; Rajendran, Surendran; Lee, James C.; Matthews, Kathleen S.

doi:10.1021/bi00171a025

Cited by 23 publications

(20 citation statements)

References 48 publications

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“…4, lane 2). This is consistent with the fact that R3 (defect in tetramerization) maintains its binding to lacO, whereas LacI-LacI interaction capability is impaired (44,45). Hence, the binding of LacI at the AatII site alone must be responsible for the basal level effect (the 2-fold effect on LacI-mediated gene silencing relief).…”

Section: Leuo-leuo Interaction-dependent Leuo Protein Binding Cooperasupporting

confidence: 74%

LeuO Protein Delimits the Transcriptionally Active and Repressive Domains on the Bacterial Chromosome

Chen

2005

Journal of Biological Chemistry

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Section: Leuo-leuo Interaction-dependent Leuo Protein Binding Cooperasupporting

confidence: 74%

LeuO Protein Delimits the Transcriptionally Active and Repressive Domains on the Bacterial Chromosome

Chen

2005

Journal of Biological Chemistry

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“…Moreover, it should be noted that c contains 20 lysine residues, many of which may be capable of reacting to give rise to different combinations of cross-links. We cannot, however, rule out the possibility that these forms reflect different structural dimers of repressor (Chen et al, 1994). Identical results to those obtained with BS 3 were obtained with disulfosuccinimidyl tartrate, a cross-linking agent with a spacer arm length of 6.4 Å (data not shown).…”

Section: Interactions Between Repressor and The Entire Mu Operatormentioning

confidence: 56%

Interactions between the Repressor and the Early Operator Region of Bacteriophage Mu

Rousseau¹,

Bétermier²,

Chandler

et al. 1996

Journal of Biological Chemistry

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The repressor of bacteriophage Mu, c, binds to three operator sites, O1, O2, and O3, overlapping two divergent promoters, which regulate the lytic and lysogenic pathways. Its binding to this operator region generates several complexes, which were analyzed by DNase I protection experiments. We demonstrate that c first binds to two 11-base pair partially repeated sequences in O2 that could represent "core" binding sites for the repressor. This initial interaction serves as an organizer of a more complex nucleoprotein structure in which O2, O1, and O3 become successively occupied. The quaternary structure of the repressor was also investigated. Size exclusion chromatography and protein-protein crosslinking experiments with chemicals that possess linking arms of various lengths indicate that the repressor oligomerizes in solution. A model is proposed describing the successive interactions of c with the operator sites O2, O1, and O3 leading to the elaboration of a higher order structure in which the early lytic functions are repressed.The repressor, c, of the temperate bacteriophage Mu controls the choice between lysogeny and lytic development. The decision is exercised at the level of transcription by binding of c to the early operator region located about 1 kilobase from the left end of the phage genome ( Fig. 1). This operator is contained within a 200-bp 1 region composed of two divergent promoters (Pe and Pc) and three operator sequences, O1, O2, and O3. These have been defined by DNase I protection of the region by the repressor (Krause et al., 1983(Krause et al., , 1986Alazard et al., 1992;Goosen et al., 1984). Binding of c to O1 and O2 appears to occur simultaneously at low repressor concentrations and prevents the initiation of transcription at the early promoter Pe. Binding to O3, which requires higher concentrations of c, inhibits transcription of the c gene from the Pc promoter (Goosen et al., 1984;Alazard et al., 1992). A binding site for integration host factor (IHF) is located between O1 and O2. This DNA-binding protein bends DNA and promotes the formation of looped structures (Craig and Nash, 1984;Gamas et al., 1987;Prentki et al., 1987;Stenzel et al., 1987;Robertson et al., 1988;Thompson et al., 1988). IHF plays a complex role in the regulation of the early functions of Mu. It has been demonstrated that it stimulates transcription from Pe, favoring lytic growth (Krause et al., 1986), possibly by direct interaction between bound IHF and RNA polymerase (Goosen and . On the other hand, IHF stabilizes the interaction among the repressor and O1 and O2 and allows the establishment and the maintenance of lysogeny (Vogel et al., 1991;Alazard et al., 1992;Gama et al., 1992;Bétermier et al., 1995).Previous results (Alazard et al., 1992) show that the interaction of c with a linear operator fragment leads to the formation of at least five high molecular weight DNA-protein complexes, which can be separated using gel retardation techniques. The complexes with lower apparent molecular weight can be chased successiv...

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“…Matthews and colleagues have argued that this C-terminal sequence forms a leucine zipper rather than a four-helix bundle in the context of native Lac repressor. Chen et al (1994) show that lengthening the tetramerization domain of Lac repressor by inclusion of l or 2 heptads from the GCN4 coiled-coil sequence helps to stabilize dimerization of Lac repressor in the background of a Y282D mutant (which knocks out the dimerization domain). Because GCN4 forms a two-chain coiled-coil structure, it is not surprising that Lac repressor should favor dimerization.…”

Section: ~ ~_ _mentioning

confidence: 97%

Characterization of a new four‐chain coiled‐coil: Influence of chain length on stability

et al. 1995

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Limited information is available on inherent stabilities of four-chain coiled-coils. We have developed a model system to study this folding motif using synthetic peptides derived from sequences contained in the tetramerization domain of Lac repressor. These peptides are tetrameric as judged by both gel filtration and sedimentation equilibrium and the tetramers are fully helical as determined by CD. The four-chain coiled-coils are well folded as judged by the cooperativity of thermal unfolding and by the extent of dispersion in aliphatic chemical shifts seen in NMR spectra. In addition, we measured the chain length dependence of this four-chain coiled-coil. To this end, we developed a general procedure for nonlinear curve fitting of denaturation data in oligomeric systems. The dissociation constants for bundles that contain a-helical chains 21, 28, and 35 amino acids in length are 3.1 x 6.7 X and 1.0 x M3, respectively. This corresponds to tetramer stabilities (in terms of the peptide monomer concentration) of 180 pM, 51 nM, and 280 f M , respectively. Finally, we discuss the rules governing coiledcoil formation in light of the work presented here.

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Construction of A Dimeric Repressor: Dissection of Subunit Interfaces in Lac Repressor

Cited by 23 publications

References 48 publications

LeuO Protein Delimits the Transcriptionally Active and Repressive Domains on the Bacterial Chromosome

LeuO Protein Delimits the Transcriptionally Active and Repressive Domains on the Bacterial Chromosome

Interactions between the Repressor and the Early Operator Region of Bacteriophage Mu

Characterization of a new four‐chain coiled‐coil: Influence of chain length on stability

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