Four dimers of λ repressor bound to two suitably spaced pairs of λ operators form octamers and DNA loops over large distances

Révet, Bernard; Wilcken‐Bergmann, Brigitte von; Bessert, Heike; Barker, Andrew; Müller‐Hill, Benno

doi:10.1016/s0960-9822(99)80069-4

Cited by 119 publications

(118 citation statements)

References 27 publications

Supporting

Mentioning

108

Contrasting

Order By: Relevance

“…Mutations (vir mutants) in one or two of the inverted repeats at p R diminishes overall binding affinity, yet the same retarded complex is observed (6). We also have preliminary evidence that CI bound at p R can interact with CI bound at the flanking sites, 1 similar to the looping observed between CI bound at the O R and O L operators (41). Taken together, these points suggest that cooperativity between DNA bound dimers of CI may be important for the existence of a stable lysogenic state.…”

Section: Discussionsupporting

confidence: 66%

The Helix-Turn-Helix Motif of the Coliphage 186 Immunity Repressor Binds to Two Distinct Recognition Sequences

Shearwin

Dodd

Egan

2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

The CI protein of coliphage 186 is responsible for maintaining the stable lysogenic state. To do this CI must recognize two distinct DNA sequences, termed A type sites and B type sites. Here we investigate whether CI contains two separate DNA binding motifs or whether CI has one motif that recognizes two different operator sequences. Sequence alignment with 186-like repressors predicts an N-terminal helix-turn-helix (HTH) motif, albeit with poor homology to a large master set of such motifs. The domain structure of CI was investigated by linker insertion mutagenesis and limited proteolysis. CI consists of an N-terminal domain, which weakly dimerizes and binds both A and B type sequences, and a C-terminal domain, which associates to octamers but is unable to bind DNA. A fusion protein consisting of the 186 N-terminal domain and the phage oligomerization domain binds A and B type sequences more efficiently than the isolated 186 CI N-terminal domain, hence the 186 C-terminal domain likely mediates oligomerization and cooperativity. Site-directed mutation of the putative 186 HTH motif eliminates binding to both A and B type sites, supporting the idea that binding to the two distinct DNA sequences is mediated by a variant HTH motif.DNA binding proteins are often modular in structure, with separate domains responsible for binding and oligomerization. Such an arrangement, even in a simple system such as the lysis-lysogeny switch of coliphage , permits remarkable control of gene expression through a series of thermodynamically linked protein-protein and protein-DNA interactions. The genetic switch, one of the most intensively studied systems in biology, has in many ways provided the basis for the study of switch biology in higher organisms (1, 2). Coliphage 186, a member of the P2 family of phage, shows essentially no similarity with at the protein or DNA level, and so the two have presumably evolved independently of each other. Nevertheless, the lysis-lysogeny switches of each show superficial similarity, and it is expected that a comparative analysis of the differences in detail between the two will improve overall understanding of switch operation, hence the present structure-function study of 186 CI repressor.The immunity repressor, CI, of coliphage 186 is responsible for maintenance of the stable lysogenic state and achieves this by binding directly over and repressing two promoters: p R , the promoter of the early lytic operon and p B , the promoter for the late promoter activator gene B (Fig. 1). 186 contains a total of four binding sites for CI, including two sites (F L and F R ) that flank the lytic promoter (6). These flanking sites play a role fine-tuning CI-regulation of transcription from p R and from the lysogenic promoter p L .1 The flanking sites F L and F R each consist of an inverted repeat, while the CI binding site at the p B promoter contains a pair of inverted repeats. These four inverted repeats share sequence similarity and are separated in each case by a five-base pair A/T-rich spacer. These CI ...

show abstract

Section: Discussionsupporting

confidence: 66%

The Helix-Turn-Helix Motif of the Coliphage 186 Immunity Repressor Binds to Two Distinct Recognition Sequences

Shearwin

Dodd

Egan

2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The cooperativity between CI 2 dimers and between Cro 2 dimers on neighboring sites is included. In addition, the enhanced CI synthesis when O R 2 is occupied by CI dimer (currently understood to be due to the looping effect between O L and O R ) is also included (37,38). The self repression of CI 2 at high concentration is also included.…”

Section: Methodsmentioning

confidence: 99%

Probability landscape of heritable and robust epigenetic state of lysogeny in phage lambda

Cao

Liang

2010

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

View full text Add to dashboard Cite

Computational studies of biological networks can help to identify components and wirings responsible for observed phenotypes. However, studying stochastic networks controlling many biological processes is challenging. Similar to Schrödinger's equation in quantum mechanics, the chemical master equation (CME) provides a basic framework for understanding stochastic networks. However, except for simple problems, the CME cannot be solved analytically. Here we use a method called discrete chemical master equation (dCME) to compute directly the full steady-state probability landscape of the lysogeny maintenance network in phage lambda from its CME. Results show that wild-type phage lambda can maintain a constant level of repressor over a wide range of repressor degradation rate and is stable against UV irradiation, ensuring heritability of the lysogenic state. Furthermore, it can switch efficiently to the lytic state once repressor degradation increases past a high threshold by a small amount. We find that beyond bistability and nonlinear dimerization, cooperativity between repressors bound to O R 1 and O R 2 is required for stable and heritable epigenetic state of lysogeny that can switch efficiently. Mutants of phage lambda lack stability and do not possess a high threshold. Instead, they are leaky and respond to gradual changes in degradation rate. Our computation faithfully reproduces the hair triggers for UVinduced lysis observed in mutants and the limitation in robustness against mutations. The landscape approach computed from dCME is general and can be applied to study broad issues in systems biology.B acteriophage lambda is a virus that infects Escherichia coli cells. It has served as a model system for studying regulatory networks and for engineering gene circuits (1-5). Of central importance is the molecular circuitry that controls phage lambda to choose between two productive modes of development, namely, the lysogenic state and the lytic state (Fig 1A). In the lysogenic state, phage lambda represses its developmental function, integrates its DNA into the chromosome of the host E. coli bacterium, and is replicated in cell cycles for potentially many generations. When threatening DNA damage occurs, phage lambda switches from the epigenetic state of lysogeny to the lytic state and undergoes massive replications in a single cell cycle, releases 50-100 progeny phages upon lysis of the E. coli cell. This switching process is called prophage induction (5).The molecular network that controls the choice between these two different physiological states has been studied extensively during the past 40 y (5-9). All of the major molecular components of the network have been identified, binding constants and reaction rates characterized, and there is a good experimental understanding of the general mechanism of the molecular switch (5). Theoretical studies have also contributed to the illumination of the central role of stochasticity (3) and the stability of lysogen against spontaneous switching (4, 10). With the advent o...

show abstract

“…This model was recently modified on the basis of physical and genetic experiments showing that a CI tetramer cooperatively bound to O R 1 ∼ O R 2 interacts with a tetramer cooperatively bound to O L 1 ∼ O L 2, located 2.3 kbp away (18)(19)(20)(21), resulting in the looping out of the intervening DNA, as shown by electron and atomic force microscopy (18,22,23). DNA loop was also observed by cooperative interactions of CI at sites separated by only five and six helical turns (23,24).…”

mentioning

confidence: 99%

Multilevel autoregulation of λ repressor protein CI by DNA looping in vitro

Lewis

Zurla

et al. 2011

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

View full text Add to dashboard Cite

The prophage state of bacteriophage λ is extremely stable and is maintained by a highly regulated level of λ repressor protein, CI, which represses lytic functions. CI regulates its own synthesis in a lysogen by activating and repressing its promoter, P RM . CI participates in long-range interactions involving two regions of widely separated operator sites by generating a loop in the intervening DNA. We investigated the roles of each individual site under conditions that permitted DNA loop formation by using in vitro transcription assays for the first time on supercoiled DNA that mimics in vivo situation. We confirmed that DNA loops generated by oligomerization of CI bound to its operators influence the autoactivation and autorepression of P RM regulation. We additionally report that different configurations of DNA loops are central to this regulation-one configuration further enhances autoactivation and another is essential for autorepression of P RM .activation | cooperativity | repression B acteriophage lambda (λ) of Escherichia coli can grow either in a lytic or lysogenic mode. In a lysogenic cell, the phageencoded λ repressor protein (CI) prevents lytic growth by directly repressing two promoters needed to express lytic functions, P L and P R (1-3). Each promoter is associated with a CI recognition site or operator,. O R is associated with promoter P RM (promoter for maintenance of repressor synthesis), which directs transcription of the cI gene in the prophage state (4-6). Each subsite binds a CI dimer (7).The CI protein autoregulates its synthesis. At low cellular CI concentration, CI enhances its own synthesis from P RM ; when high, CI represses P RM (1,8,9). It was originally believed that both positive and negative autoregulations are achieved exclusively by the action of CI dimers at the P RM -O R -P R sequence of the phage genome (1, 10) (Fig. 1A), based on the following observations. (i) There is a hierarchy of intrinsic binding affinities of a CI dimer to individual operators sites: (11)(12)(13)(14)(15)(16)(17); (ii) CI bound to the intrinsically weak O R 2 site is strengthened by cooperative interactions with CI bound to the stronger adjacent O R 1 site, and the ensemble of two CI dimers at the O R 1 ∼ O R 2 sites represses P R and activates P RM (13, 16) (Fig. 1A); (iii) at high CI concentrations, a CI dimer can bind to the weakest operator site, O R 3, repressing P RM (1, 2) (Fig. 1C). Incidentally, a second pair of CI dimers binds cooperatively to O L 1 ∼ O L 2, and represses P L (7).This model was recently modified on the basis of physical and genetic experiments showing that a CI tetramer cooperatively bound to O R 1 ∼ O R 2 interacts with a tetramer cooperatively bound to O L 1 ∼ O L 2, located 2.3 kbp away (18-21), resulting in the looping out of the intervening DNA, as shown by electron and atomic force microscopy (18,22,23). DNA loop was also observed by cooperative interactions of CI at sites separated by only five and six helical turns (23, 24). The kinetic and thermodynamic properties ...

show abstract

Four dimers of λ repressor bound to two suitably spaced pairs of λ operators form octamers and DNA loops over large distances

Cited by 119 publications

References 27 publications

The Helix-Turn-Helix Motif of the Coliphage 186 Immunity Repressor Binds to Two Distinct Recognition Sequences

The Helix-Turn-Helix Motif of the Coliphage 186 Immunity Repressor Binds to Two Distinct Recognition Sequences

Probability landscape of heritable and robust epigenetic state of lysogeny in phage lambda

Multilevel autoregulation of λ repressor protein CI by DNA looping in vitro

Contact Info

Product

Resources

About