Kinetic analysis of mutations affecting the cII activation site at the PRE promoter of bacteriophage lambda.

Shih, Ming-Che; Gussin, Gary N.

doi:10.1073/pnas.81.20.6432

Cited by 11 publications

(4 citation statements)

References 26 publications

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“…In a cell following the lysogenic pathway, CI is initially expressed from the strong promoter P RE , rather than P RM (24). P RE is regulated by the phage CII protein (24,25). We expect the level of CI provided by P RE to be approximately the same in wild type and JL516.…”

Section: Discussionmentioning

confidence: 99%

Cooperative DNA binding by CI repressor is dispensable in a phage λ variant

Babić

Little

2007

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

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Complex gene regulatory circuits contain many interacting components. In principle, all of these components and interactions may be essential to the function of the circuit. Alternatively, some of them may be refinements to a simpler version of the circuit that improve its fitness. In this work, we have tested whether a particular property of a critical regulatory protein, CI, is essential to the behavior of the phage regulatory circuit. In the lysogenic state, CI represses the expression of the lytic genes, allowing a stable lysogenic state, by binding cooperatively to six operators. A mutant phage lacking cooperativity because of a change in cI could not form stable lysogens; however, this defect could be suppressed by the addition of mutations that altered two cis-acting sites but did not restore cooperativity. The resulting triple mutant was able to grow lytically, form stable single lysogens, and switch to lytic growth upon prophage induction, showing a threshold response in switching similar to that of wild-type . We conclude that cooperative DNA binding by CI is not essential for these properties of the circuitry, provided that suppressors increase the level of CI. Unlike wild-type lysogens, mutant lysogens were somewhat unstable under certain growth conditions. We surmise that cooperativity is a refinement to a more basic circuit, and that it affords increased stability to the lysogenic state in response to environmental variations.cooperativity ͉ evolution of regulatory circuitry ͉ gene regulatory circuit ͉ systems biology ͉ prophage induction C omplex gene regulatory circuits include many components and interactions. It is likely that these features are advantageous and have been selected for during the course of evolution. However, certain features may be refinements to the system rather than essential to its operation. In this view, a complex system might have arisen in a much simpler form, with refinements being added later as the system evolved toward its present form. This idea predicts that existing circuits might tolerate the removal of certain features (perhaps with the addition of suppressors) and still show qualitatively normal behavior. In this work, we have tested this prediction in a well characterized complex circuit, the bistable switch of phage , by removing one feature long believed to be essential for its proper operation.A cell infected with can follow either of two pathways, the lytic or lysogenic pathways. In the lytic pathway, the phage DNA replicates, and Ϸ100 new virions are made and released upon cell lysis. In the lysogenic pathway, the viral genome integrates into the host chromosome, and the lytic genes are repressed by the action of CI protein. This pathway leads to a stable regulatory state, the lysogenic state. In this state, CI binds tightly to two operators in the O R region, O R 1 and O R 2, repressing the expression of the lytic P R promoter and stimulating its own expression from another promoter, P RM (1).Although the lysogenic state is highly stable and can be main...

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Section: Discussionmentioning

confidence: 99%

Cooperative DNA binding by CI repressor is dispensable in a phage λ variant

Babić

Little

2007

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

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“…In vitro transcriptions. The runoff transcription assay was performed in 25 l of the same transcription buffer used in the footprinting experiments with the addition of 50 g of heparin per ml and 200 M nucleoside triphosphates, following published procedures (22), except that dithiothreitol was found to inhibit MetR activation and therefore was omitted. Template DNA (final concentration, 2 to 3 nM) was allowed to bind to MetR protein in the presence or absence of 2 mM D,L-homocysteine.…”

Section: Methodsmentioning

confidence: 99%

Characterization of a second MetR-binding site in the metE metR regulatory region of Salmonella typhimurium

Urbanowski

Stauffer

1995

J Bacteriol

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Transcription of the metE gene in Salmonella typhimurium and Escherichia coli is positively regulated by the MetR protein, with homocysteine serving as a coactivator. It was shown previously that MetR binds to and protects from DNase I digestion a 24-bp sequence in the metE metR regulatory region from nucleotides ؊48 to ؊71 relative to the metE transcription initiation site (designated as site 1). In this study, we show that purified MetR protein also binds to and protects a second 24-bp sequence adjacent to the original site, from nucleotides ؊24 to ؊47 relative to the metE transcription initiation site (designated as site 2). Single and multiple base changes were introduced into sites 1 and 2 in a metE-lacZ fusion. Base pair changes in site 1 or site 2 away from the MetR consensus binding sequence resulted in decreased metE-lacZ expression, suggesting that both sites are necessary for expression. DNase I footprint analysis showed that MetR bound at the high-affinity site 1 enhances MetR binding at the low-affinity site 2. A 2-bp change in site 2 toward the MetR consensus binding sequence resulted in high metE-lacZ expression; the increased expression was MetR dependent but homocysteine independent.Regulation of the genes that make up the met regulon of both Salmonella typhimurium and Escherichia coli are under positive and/or negative control (17,18). Transcription of all the met genes, except metH, is negatively regulated by the MetJ repressor, with S-adenosylmethionine serving as a corepressor (17,18). Transcription of metE (3,14,29), metA (12), metF (7), and metH (3, 14, 29) requires an activator, the MetR protein, for full expression. The pathway intermediate homocysteine serves as a coregulator for MetR-mediated regulation of these genes and has a negative effect on metA and metH expression (4, 12, 28) and a positive effect on metE and metF expression (4, 6, 28). Homocysteine is the end product of the reactions catalyzed by the metA, metB, and metC gene products in the nonfolate branch of the pathway and serves as the acceptor for the methyl group donated by the folate branch of the pathway. The role of homocysteine as coregulator for MetR-mediated activation may thus serve to balance the two branches of the methionine pathway.The promoters for the metE and metR genes overlap and are divergently transcribed from a shared regulatory region (16) (Fig. 1). Previous genetic and biochemical data identified a binding site for MetR protein in the metE metR regulatory region (site 1 in Fig. 1) that is necessary for MetR-mediated activation of the metE gene (27). We show here that a second MetR-binding site, with a lower affinity for MetR protein than that of site 1, is located adjacent to site 1 and is also required for MetR-mediated activation of the metE gene (designated as site 2 in Fig. 1). A possible model for these two binding sites in MetR-mediated activation of the metE gene is discussed. MATERIALS AND METHODSBacterial strains and plasmids. Strain GS760 is metB1 metJ97 pheA905 thi ⌬lacU169 ara⌬129 rpsL, s...

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“…Presumably, other CII or CII-like proteins from lambdoid phages also have a closely similar sequence for CII binding. It has been shown that the change of one base in either of the tetrad repeats, although resulting in reduced CII binding and impaired lysogenicity, may be tolerated (43). The C1 protein of P22 (which is analogous to CII) recognizes the T-T-G-C-N 6 -T-T-G-T sequence present in p E of P22 (44,45).…”

Section: Resultsmentioning

confidence: 99%

Structure of λ CII: Implications for recognition of direct-repeat DNA by an unusual tetrameric organization

Datta

Panjikar

Weiss

et al. 2005

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

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The temperate coliphage , after infecting its host bacterium Escherichia coli, can develop either along the lytic or the lysogenic pathway. Crucial to the lysis͞lysogeny decision is the homotetrameric transcription-activator protein CII (4 ؋ 11 kDa) of the phage that binds to a unique direct-repeat sequence T-T-G-C-N 6-T-T-G-C at each of the three phage promoters it activates: pE, pI, and paQ. Several regions of CII have been identified for its various functions (DNA binding, oligomerization, and susceptibility to host protease), but the crystal structure of the protein long remained elusive. Here, we present the three-dimensional structure of CII at 2.6-Å resolution. The CII monomer is comprised of four ␣ helices and a disordered C terminus. The first three helices (␣1-␣3) form a compact domain, whereas the fourth helix (␣4) protrudes in different orientations in each subunit. A four-helix bundle, formed by ␣4 from each subunit, holds the tetramer. The quaternary structure can be described as a dimer of dimers, but the tetramer does not exhibit a closed symmetry. This unusual quaternary arrangement allows the placement of the helix-turn-helix motifs of two of the four CII subunits for interaction with successive major grooves of B-DNA, from one face of DNA. This structure provides a simple explanation for how a homotetrameric protein may recognize a direct-repeat DNA sequence rather than the invertedrepeat sequences of most prokaryotic activators.direct-repeat recognition ͉ helix-turn-helix motif ͉ transcription activator T he CII protein of phage is a transcription activator that has attracted the attention of researchers for diverse reasons. On one hand, CII is a key regulator for the temperate coliphage, enabling the virus to make the choice between the lytic and lysogenic pathways of development (1). On the other hand, CII is the only known example of a DNA-binding protein that recognizes direct-repeat sequences in DNA (2) rather than the commonly observed inverted-repeat sequences in most prokaryotic transcription activators (3).After infecting its host bacterium Escherichia coli, can follow either of its two alternate modes of development, namely, lytic or lysogenic. The choice between these pathways is an important decision in the life cycle of the phage (1, 4) and is influenced by factors such as temperature, nutrient concentration, and multiplicity of infection. In fact, this decision-making process was one of the early paradigms for studying gene control and the understanding of molecular switches (5). The phage has developed an intricate molecular machinery for this purpose, involving several proteins from both the phage and the bacterial host (1). Lysogenic development requires the synthesis of CI repressor, for which CII is absolutely essential. CII coordinately activates transcription from the three phage promoters p E (required for initial synthesis of repressor CI), p I (necessary for synthesizing the integrase protein), and p aQ (directs the synthesis of an antisense RNA to reduce late gene exp...

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Kinetic analysis of mutations affecting the cII activation site at the PRE promoter of bacteriophage lambda.

Cited by 11 publications

References 26 publications

Cooperative DNA binding by CI repressor is dispensable in a phage λ variant

Cooperative DNA binding by CI repressor is dispensable in a phage λ variant

Characterization of a second MetR-binding site in the metE metR regulatory region of Salmonella typhimurium

Structure of λ CII: Implications for recognition of direct-repeat DNA by an unusual tetrameric organization

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