Model for lactose repressor protein and its interaction with ligands.

Dunaway, M; Manly, Susan P.; Matthews, Kathleen S.

doi:10.1073/pnas.77.12.7181

Cited by 27 publications

(10 citation statements)

References 49 publications

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“…These data extend the physical evidence for the specific recognition and binding of operator DNA by the trypsinresistant core of lac repressor (22). Further, these results corroborate the importance of the inner asymmetric region of the operator sequence in the specific interaction with the protein (37) (Fig. 4) …”

Section: Methodssupporting

confidence: 75%

Perturbation of lac operator DNA modification by tryptic core protein from lac repressor.

Manly¹,

Bennett²,

Matthews³

1983

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

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Operator DNA fragments were modified in the presence of lac repressor protein or its trypsin-resistant core. Operator DNA was alkylated or cleaved enzymatically with these related proteins present to compare the influences of their binding on the reactivities or enzymatic susceptibilities of individual bases in the sequence. These two protein species have pronounced and distinguishable effects on the reactivity of the bases of the operator fragment toward methylation by dimethyl sulfate. Perturbation of base alkylation by the trypsin-resistant core repressor is most pronounced in the inner, asymmetric region of the operator DNA, while repressor effects extend further on either end of the operator sequence. Digestion of the two protein-operator complexes by DNase I yields fragment patterns that differ primarily in extent of protection. These data extend the experimental base supporting the involvement of the core region of the lac repressor in addition to its NH2 termini in the operator-specific binding activity of this protein.Repression of transcription of the genes coding for enzymes involved in lactose metabolism in Escherichia coli is the result of the specific, tight binding of lac repressor to its target site, the operator (1). Insight into how the lactose repressor binds, including the molecular details of its specific DNA contacts, is important in understanding interactions affecting gene expression. The affinity of the lac repressor is modulated by binding to small sugars. Binding to certain of these sugars, inducers, causes a conformational change in the protein that reduces the affinity of binding to the operator and thereby allows the excess of "nonspecific" DNA binding sites in the genome to compete effectively with the operator for the protein (2). Read-through of the operator by RNA polymerase then allows the transcription of the lac messages. Understanding how the operator and inducer binding sites are related in terms of protein topology is central to deciphering the mechanism of repression/induction.Genetic analysis of lad mutants coupled with physical characterization of some mutant repressors suggested that the sugar effector and DNA binding activities had few, if any, common determinants (3-9). Mutants aberrant in DNA binding mapped predominately in the NH2-terminal 60 amino acids of repressor. Assembly mutations were found close to the COOH terminus, especially residues 250-280. Mutations affecting inducer binding were found exclusively in the region 60-360 (none were located in the NH2-terminal region) and mapped principally in the region spanning residues 200-250. A "hinge" or "transmitter" region consisting of amino acids 50-80 was postulated to convey the presence of inducer to the operator site (7,8).Initial studies using mild tryptic cleavage yielded two fragments of repressor: NH2-terminal fragments (1-51 or 1-59) and tetrameric core repressors (52-360 or 60-360) (10-12). The activities associated with these fragments supported the view of segregation of activities obtained ...

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Section: Methodssupporting

confidence: 75%

Perturbation of lac operator DNA modification by tryptic core protein from lac repressor.

Manly¹,

Bennett²,

Matthews³

1983

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

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“…Repressor dimers may similarly bind simultaneously to the adjacent operator sites OR2 and OR3 in a cooperative manner (6); the free energy of this cooperative interaction is AG23. 4. Cooperative interaction between adjacently bound repressors at OR2 and OR3 occurs only when OR1 is vacant.…”

Section: The Systemmentioning

confidence: 99%

“…Models for interactions at the lac operon have been developed to include effects of inducer and nonspecific DNA on the binding of lac repressor (2)(3)(4). The systems of A and other inducible phages differ from lac by using multiple operator binding sites that have cooperative interactions between bound repressors (1).…”

mentioning

confidence: 99%

Quantitative model for gene regulation by lambda phage repressor.

Ackers¹,

Johnson²,

Shea³

1982

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

575

602

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A statistical thermodynamic model has been developed In prokaryotes, genes are commonly switched on and off by the interactions of regulatory proteins with specific DNA sequences. A particularly complex example of such a switch is found at the right operator (OR) ofbacteriophage A: this operator consists of three tandem DNA sites that are recognized by two phage-encoded regulatory proteins (the A repressor and cro protein). When phage A is in the "lysogenic state," the A repressor (cI gene product) is synthesized and occupies sites OR1 and OR2 In this configuration of OR, the cro gene is repressed and the ci gene is transcribed.The phage switches into the "lytic state" when the repressor protein is cleaved in half by the recA protein, an action initiated by DNA damage. As repressor is destroyed, the cro gene is derepressed and the cro protein is made and occupies site OR3, turning off transcription of the cl gene, yet allowing its own synthesis. In this fashion, the phage can switch from one state (lysogeny) to another (lytic growth) in response to an external signal (for review, see ref. 1).In this paper, we consider the lysogenic state (repressor on, cro off) in an attempt to understand the physical principles that govern the ways in which the protein-DNA and protein-protein interactions operate in concert to produce the known physiological behavior. We show that a model based on statistical thermodynamic assumptions is sufficient to account for some of the known physiological properties of the repressor-operator regulatory system. A brief summary of certain aspects of this work has been presented elsewhere (1).Models for interactions at the lac operon have been developed to include effects of inducer and nonspecific DNA on the binding of lac repressor (2-4). The systems of A and other inducible phages differ from lac by using multiple operator binding sites that have cooperative interactions between bound repressors (1). This requires a more elaborate theoretical approach to the protein-DNA binding problem-one that has not previously been developed. The mathematical model we present here incorporates a set of rules and assumptions derived from previous genetic, biochemical, and structural studies. Combination of this information with statistical thermodynamic assumptions generates a quantitative formulation that incorporates salient features of the qualitative description developed over the last several years (1,(5)(6)(7)(8)(9)). This quantitative model has the following significance. (i) It provides a way to test assumptions regarding the physical bases for operation of the system. Thus, it predicts quantitatively the activities of the two OR-controlled promoters (PR and PRM) as a function of repressor concentration, and these predictions can then be compared with known.physiological properties of the system. (ii) It points out features of the A operator system that were not obvious (or at least fully appreciated) from previous experiments. (iii) It incorporates a theory for interpreting cooperati...

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“…We expected that the nprA gene product was the transcriptional activator of the nprS promoter, as has been shown for the other transcriptional activators (22). Actually, the 28-bp palindromic sequence, which was similar to the targets of general transcriptional regulators (LacI [3], cyclic AMP receptor protein [1], DeoR [29], etc. ), was found 5 bp upstream of the nprS promoter, and the sequence might be used as a regulatory region.…”

Section: Resultsmentioning

confidence: 99%

Cloning and nucleotide sequences of the Bacillus stearothermophilus neutral protease gene and its transcriptional activator gene

Nishiya

Imanaka

1990

J Bacteriol

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Both the neutral protease gene (nprS) and its transcriptional activator gene (nprA) from Bacillus stearothermophilus TELNE were cloned in Bacillus subtilis by using pTB53 as a vector plasmid. The presence of the nprA gene enhanced protease synthesis by about fivefold. The nucleotide sequences of nprS and its flanking regions were determined. nprS was composed of 1,653 base pairs and 551 amino acid residues. A Shine-Dalgarno (SD) sequence was found 9 bases upstream from the translation start site (ATG). The deduced amino acid sequence was very similar to that of another thermostable neutral protease gene, nprM (M. Kubo and T. Imanaka, J. Gen. Microbiol. 134:1883Microbiol. 134: -1892Microbiol. 134: , 1988. The amino acid sequence of the extracellular neutral protease NprS was completely identical to that of NprM. By deletion analysis and substitution of the original promoter with a foreign promoter, it was found that the nprA gene existed upstream of nprS. It was also found that a possible target region (palindromic sequence) of the gene product of nprA existed near the promoter sequence of nprS. The nucleotide sequences of nprA and its flanking regions were determined. The DNA sequence revealed only one large open reading frame, composed of 1,218 base pairs (406 amino acids; molecular weight, 49,097). The SD sequence was found 4 bases upstream from the translation start site (GTG). A possible promoter sequence (TTGAAG for the -35 region and AATTTT for the -10 region) was also found about 20 bases upstream of the SD sequence. The nprA gene was separated from nprS by a typical terminator sequence. By constructing an in-frame fusion between the lacZ gene and the 5' region of the nprA gene, it was demonstrated that the coding region of nprA was indeed translated in vivo. Three palindromic sequences, which were highly homologous with a possible target region by NprA, were also found in the 5' region of the nprA gene. This suggests that the expression of nprA is autoregulated. From the time course of the production of NprA-LacZ fusion protein, it was indicated that nprA was expressed in late log phase, whereas nprS was expressed in the stationary phase. The NprA protein had consensus regions homologous to the DNA recognition domains of DNA-binding proteins but showed no sequence homology with any other regulatory proteins for protease production. It is inferred that NprA protein binds to the upstream region of nprS promoter and activates transcription of nprS. A new regulatory mechanism by the nprA-nprS genes is discussed.

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Model for lactose repressor protein and its interaction with ligands.

Cited by 27 publications

References 49 publications

Perturbation of lac operator DNA modification by tryptic core protein from lac repressor.

Perturbation of lac operator DNA modification by tryptic core protein from lac repressor.

Quantitative model for gene regulation by lambda phage repressor.

Cloning and nucleotide sequences of the Bacillus stearothermophilus neutral protease gene and its transcriptional activator gene

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