1. Volume 21, Issue 5, Pages 605-615 (March 2006)
The Structural Basis of Cooperative Regulation at an Alternate Genetic Switch 
Heather W. Pinkett, Keith E. Shearwin, Steven Stayrook, Ian B. Dodd, Tom Burr, Ann Hochschild, J. Barry Egan, Mitchell Lewis 
Molecular Cell 
Volume 21, Issue 5, Pages (March 2006)
DOI: /j.molcel
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2. Figure 1 Comparison of the λ and 186 Switch Regions
(A) Organization of the λ genetic switch. Genes are indicated as boxes, promoters as arrowheads, transcripts as solid lines, and terminators as stem loops. The OR and OL regions are enlarged to show the positions of the λ CI operators (represented as green arrows). Note that the PR and PRM promoters are arranged back to back.
(B) Organization of the 186 genetic switch. In contrast to λ, 186 CI binds two distinct types of operator and the pR and pL promoters are arranged face to face. A type operators are shown in red, and B type operators are shown in blue. Flanking operators, FL and FR, are located 327 bp to the left and 352 bp to the right, respectively, of pR. The pR region is enlarged to show positions of CI operators. pR contains three operators, each located two turns of the helix (center to center) from its neighbor. At pL, there is a weak A type operator, located 63 bp from the rightmost pR operator.
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3. Figure 2 Structures of the CTD of λ and 186 Repressor
(A) The monomeric structures of the CTD of 186 (colored green) and λ (colored blue) illustrate the similarity in the topology. The regions that are most dissimilar are shown uncolored. In the 186 repressor CTD, there are two additional β strands that are not observed in the λ structure, whereas λ has a large loop that is not present in 186.
(B) The structure of a 186 CTD dimer and the same orientation of a λ CTD dimer illustrate the differences in dimer formation. The λ structure appears more cylindrical, whereas the 186 CTD dimer is clearly bent. Residues identified by genetic screens as disrupting dimer formation are illustrated in space-filling representation and are located at the dimer interfaces.
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4. Figure 3 Quaternary Assembly of 186 Repressor CTD
In the crystal, the CTD forms a 14-mer, arranged as a heptamer of dimers. Two dimers are highlighted with the same color scheme as in Figure 2B. In one view (left), the dimer axis is in the plane of the page and the pseudo 7-fold axis is perpendicular to the plane of the page. The 14-mer is approximately 102 Å in height, 57 Å in width, with a hollow core that has a diameter of ∼36 Å. In the second view of the 14-mer (right), the 7-fold axis is in the plane of the page and illustrates that the dimers associate to form the 14-mer. The amino acids identified in a screen for cooperativity mutants are shown in space-filling representation. For visual simplicity, the mutants were divided into two groups based upon their location in the structure. These residues map to the dimer-dimer interface, suggesting that the observed higher-order structure is biologically significant.
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5. Figure 4 Activity of 186 CI Repressor Association Mutants In Vivo
(A) All residues that were identified genetically as being important for oligomer assembly are shown in space-filling representation. Note the separation of the two classes of mutations. The positions of the D107A substitution at the monomer-monomer interface (green) and the E146K substitution at the dimer-dimer interface (purple) are indicated.
(B) Full-length 186 CI association mutants were defective in their ability to repress the 186 pR promoter in vivo. The ability of the weak monomer-monomer association mutant (D107A), the dimer-dimer cooperativity mutant (E146K), and a D107A/E146K double mutant to repress a pR:lacZ reporter gene was assayed by using the reporter strain, NK7049 (λRS45lacZΔYA-pBC2-HincII–SnaBI FL−pR+pL+FR−), which carried a chromosomally inserted single copy of the pR:lacZ fusion (Dodd and Egan, 2002), pZS-CI to supply CI or its derivatives from the plac promoter, and the pUHA-1 plasmid as a source of Lac repressor. The vector only strain carried the parental pZS45 plasmid. Each point is the mean of at least eight assays and error bars represent 95% confidence limits.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 Structure of the Full-Length 186 CI E146K Repressor
The 186 NTD is a compact unit consisting of five helices (left). The helix-turn-helix (HTH) motif is shown in red. In the crystal, the HTH motifs are in a conformation that is inconsistent with operator binding. In the absence of the operator, the orientation of the NTD appears to be governed by lattice contacts in the crystal and do not obey the same symmetry as the CTD. To the right are the DNA binding domains of 186 (pink) and λ (blue) oriented to highlight the HTH motif. The overall structures are quite similar, although the relative orientation of helix one is shifted in the two structures. However, the most notable difference is the virtual absence of helix five in 186 repressor.
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7. Figure 6
Gene Regulation at the 186 Genetic Switch by Competitive DNA Binding
(A) Model for regulation of 186 promoters by the CI 14-mer, where the location of CI operators are indicated by circles, with the weak pL site in gray. The 186 reporter profiles from Dodd and Egan (2002) illustrate the importance of the flanking sequences for regulation of the pL promoter. Species 1–6 are described in the text. According to this model, as many as six contiguous operators, centered two turns of the DNA helix apart, can bind around the circumference of the 186 CI 14-mer wheel. Modeling suggests that a seventh contiguous operator could not bind, nor could nonadjacent operators bind to adjacent CI dimers, due to steric clashes of the DNA. Noncontiguous operators, on the other hand, would not be sterically hindered from binding nonadjacent dimers within the 14-mer. The spacing from the three operators at pR to the weak operator at pL is ideal to allow binding of these operators to six of the seven dimers within the 14-mer, repressing both pR and pL (species 2). The flanking sites assist repression of pR and simultaneously increase pL activity by competing with the binding of pL to the CI 14-mer (species 3 and 4).
(B) Testing the competitive DNA binding model by using a chimeric λ-186 repressor. Regulation of λ PR and PRM by a λ-186 CI chimera was examined. The chimera consists of the λ CI NTD (residues 1–92) and the 186 CI CTD (residues 84–192). The chimeric repressor was expressed under Lac repressor control from pZC320 λN/186C-CI to the PR: and PRM:lacZ reporter constructs shown. pUHA1 was used to supply Lac repressor. Error bars are 95% confidence limits (n = 6). Reporter strains and β-galactosidase assays were as described in Dodd et al. (2001, 2004).
(C) As described in the text, OL stimulation of PRM can be explained if steric hindrance prevents nonadjacent operators from binding to adjacent CI hybrid dimers in the CI wheel. The λ NTDs are shown in dark gray, the 186 CTDs in light gray, and the positions of the λ operators are shown as circles.
Molecular Cell  , DOI: ( /j.molcel )
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