III. Intramolecular Interactions
IV. Intermolecular Interactions
A. DNA-Binding Interactions
B. Inducer Binding Interactions
C. Allosteric Signal Transduction
D. Role of the Tetramere
References
The lac operon contains a set of three genes, lacZYA, that enable cells to regulate the uptake and metabolism of Beta-galactosides, such as lactose. The lacZ gene encodes the enzyme Beta-galactosidase. Beta-galactosidase has the ability to metabolize galactosides to simple sugars, cleaving lactose into a molecule of glucose and galactose. The lacY gene encodes Beta-galactoside permease, a membrane-bound protein that transports Beta-galactosidase into the cell. The lacA gene encodes a protein called Beta-galactoside transacetylase. This enzyme transfers an acetyl group from acetyl-CoA to the Beta-galactoside.
When the lac repressor protein is bound to the promotor on the lacZYA operator, transcription of the three lac genes is inhibited. Active transcription requires the presence of an inducer molecule such as the lactose derivative allolactose. (Allolactose is made from lactose by an enzyme.) Only when an inducer is bound to the repressor molecule does a conformational change occur, and the DNA is released from the repressor molecule. This permits RNA polymerase to bind and transcribe the lac genes. The repressor protein therefore has two binding sites, one for the lac operator (a sequence overlapping the promoter) and another for the inducer molecule (allolactose).
The presence of allolactose signals the cell that lactose is available to be metabolized. As the lactose is metabolized, both lactose and allolactose eventually are used up and no longer induce the operon. To maintain induction of the lac operon in a laboratory experiment, we may use a non-metabolizable inducer such as iso-proppylthiogalactoside (IPTG). IPTG induces lacZYA expression without being used up.
There are three main domains in the lac repressor molecule. The NH2 terminal domain (residues 1-62), the coreprotein domain (residues 62-340), and the COOH-terminal domain (residues 340-357) <>. As mentioned above, the NH2 terminal domain of the lac repressor is involved with the DNA binding <>. The NH2 terminal ends can actually be subdivided into two separate functioning domains: the DNA-binding region (residues 1-45) and the hinge region (residues 46-62) <>. The DNA-binding region, also known as the repressor head-piece, contains a helix-turn- helixmotif (HTH). This motif occurs over the first two helixes of the molecule (residues 6-13, 17-24) <>. A small domain with a rich hydrophobic core is formed by the two alpha helixes in the HTH motif (residues 6-25). The hinge region links the DNA-binding region to the core of the protein. This region of the lac repressor allows the DNA-binding region and the core of the protein to move independent of one another. In the presence of DNA, the hinge region has been shown to make specific interactions with the lac operon, orienting the head-piece for DNA-binding. The hinge region is able to do so through the ordering of residues 50-58, which transform from a coil to a helix in the presence of the lac operon <>. In the absence of the lac operator DNA, the residues are "relaxed," allowing the repressor head-piece to dangle freely. The core repressor protein also has two subdomains, an NH2 and COOH terminal end <>. The core of the protein is also known as the inducer binding domain. The two subdomains of this region are very similar and are connected at three sites by the shared amino acid chain. Both subdomains have similar tertiary structures: a six stranded parallel beta sheet, surrounded by four alpha helixes <>. The COOH-terminus of the protein is responsible for the molecule being a tetramere. The tetramere is formed when the alpha helixes of the four monomers associate in an antiparallel fashion <>. Though the molecule is a tetramere, it is easier to think of it as a dimer of dimers. This is due to the skewed, V shape of the molecule. Though all of the monomers of the molecule are oriented in the same direction, the dimers are oriented in different planes, causing the V shape in the protein, with the DNA-binding region at the furthest points of the V. III. Intramolecular Interactions There are four principle clusters of amino acids in the tetramer that comprise the interface between the two monomers of each dimer <> : residues 70-100, 221- 226, 250-260, and 275-285 <>. Point mutations in any of these clusters, excluding 250-260, will result in only the monomeric form of the repressor. The binding between the two dimers is extremely strong due to the number of interactions. Tetramere formation is fairly weak because binding is a result of hydrophobic interactions between leucine residues in the COOH terminal helices of the protein. Both of the binding helices are from different dimers. The leucine heptad repeats of one COOH helix of one monomer are bound to the leucine heptad repeats of the diagnal monomer (residues 338-356) <>. It is clear that these residues are integral to tetramere formation, because mutations at these sites prevent the formation of the tetramere structure, resulting in two unattached dimers. Because of this weak interaction at the COOH end of the molecule, the lac repressor is sometimes considered a "tethered dimer." IV. Intermolecular Interactions A. DNA-Binding Interactions Definite information has yet to be gathered concerning the exact DNA binding interactions between the repressor head-piece <> and the operator sequence of the lac gene. The repressor tetramere is bound to two independent and symmetric operator DNA double helices. The conformation between the two helices in the HTH motif, discussed above, changes upon DNA binding. Residues Asn-25 and His-29 <> have been shown to come in contact with the DNA upon binding. Slipjer et al (1997), have shown that there is a great decrease in the flexibility of the loop joining the two HTH helices due to binding interactions between His-29 of the monomer and Thy-3 of the operator DNA <>. In addition, the residues Gln-18 and Arg-22, have also been shown to have mobility constrict upon repressor binding to DNA, indicating that the two are most likely involved with DNA binding <>. The interactions above occur in the major groove of the DNA. It has also been shown that the leucine residues at position 56 (from each monomer), makes direct contact with the minor groove of the DNA <>. These leucine residues bind close to the center of the operator DNA and act as a lever, prying open the minor groove of the DNA. In addition to being pried open upon repressor binding, the minor groove also decreases in size. The decrease in the size of the minor groove results in the bending of the DNA which occurs when a full repressor tetramere binds to the DNA. The bend in the DNA (total of 60 A radius of curvature) is localized in the center of the operon, where there is more than a 45A turn. DNA bending The DNA fragments bound to the tetramere, are separated by about 25 angstroms. In addition, there are no interaction between the two pieces of DNA. B. Inducer Binding Interactions The inducer molecule, in this case an IPTG molecule, is bound to the lac repressor at the two protein core sub domains <>. The galactoside, IPTG is used as the inducer instead of a lactose molecule, because lactose can be degraded in the cell, while IPTG can not. Due to the IPTG molecule being semi-symmetric, it can interact with the repressor in one of two ways. The repressor has the opportunity to make three or four bonds to the hydroxyl groups of the IPTG inducer molecule. Specifically, interactions through four hydrogen bonds: Asn246, with O2 of IPTG, Arg197 with O3 and O4, and Asp149 with O6. In addition, the Trp220 amino acid is in van der walls contact with an isopropyl group of IPTG <>. There is also a hydrophobic "sugar pocket" with in the core protein, involving the amino acids: Leu73, Ala75, Pro76, Ile79, Trp220, and Phe293 <>. There are a collection of altered lac repressor molecules that can not elicit a release of the DNA upon binding. These molecules are either defective in sugar binding or at transmitting the allosteric signals to the protein binding domain. These mutant molecules are said to display the Is phenotype. Some of the mutations of these Is phenotype molecules are scattered around the molecule, however most are concentrated in five regions: residues 70-80, 90-100, 190-200, 245-250, and 272-277 <>. It is important to realize where these crippling mutations lie because these sites are integral for either inducer binding or allosteric signal transduction. Most of these sites are near the sugar binding site, with the exception of residues 90-100. For this reason, it is hypothesized that these residues are responsible for allosteric signaling. They form the first Beta sheet of the NH2 terminal subdomain of the core domain, located at the dimer interface. C. Allosteric Signal Transduction There are two different structures of the lac repressor molecule corresponding to the induced and repressed states (bound to DNA). It is thought that this change is most likely propagated through the hinge piece discussed above. The change of the NH2 subdomain, as a result of inducer binding, results in a 3.5 angstrom displacement of the alpha carbon of residue 62: this amino acid is the last amino acid of the head-piece, connecting the hinge region to the core protein molecule. The alteration of this residue, results in disruption in the interaction of the hinge helices, a freeing of the HTH bound DNA, and a reduction in the affinity for the operator by the lac repressor molecule. This is similar to the allosteric changes in the oxy form of hemoglobin. Specifically, in the induced state, there are electrostatic alterations between chains in a dimer in the following residues: Lys84 and Glu100, Gln117 and Arg118, and His 74 and Asp 278 <>. The changes in binding between these residues is a result of a 3 angstrom change in the positioning of their alpha carbon. D. Role of The Tetramere If the only allosteric changes in the repressor molecule occure between chains in a dimer, the question arises as to why there is a need for the tetramere structure at all. A single repressor molecule binds to two operators that are between 93 and 401 bp apart. One suggested mechanism is that the repressor dimers bind to separate operator sequences, forcing the DNA to conform to a loop structure. In order for this kind of DNA interaction to occur, there must be a sufficient length between the operator sequences, in addition to certain properties of the DNA. The formation of this loop structure makes DNA polymerase binding even more unfavorable than if only one repressor dimer were bound to the DNA. It is aparent that this mechanism has evolved in cells in order to completely shut off lac operon transcription, when the inducer is bound. This is most likely a result of the extreme importance of metabolism regulation in cells. Another possible mechanism for bending the DNA is to place another DNA binding protein to the DNA loop region causing a sharper bend to the loop. Cap interacts with the operator sequence between the first and second operator in order to induce a 900 bend in the DNA over 30 bp. It has been shown that the CAP repressor combination binds DNA tighter than the repressor molecule alone. This works out well because the production of CAP is increased at times in the cell when energy needs to be conserved, and lactose cleavage needs to be slowed. Chime Index References Lewis, M., Chang G., et al., 1996. "Crystal structure of teh Lactose Operon Repressor and Its Complexes with DNA and Inducer." Science 271:1247-1254. Lamerichs, R.M.J.N., et al., 1989. "H NMR study of a Comples between the lac Repressor Headpiece and a 22 Base Pair Symmetric lac Operator." Biochemistry 28:2985-2991. Friedman, Alan M., et al., 1995. "Crystal Structure of lac Repressor Core Tetramere and Its Implications for DNA looping." Science 268:1721-1727. Slijper, M. et al., 1997. "Backbone and Side Chain Dynamics of lac Repressor Headpiece (1-56) and Its Complex with DNA." Biochemistry 36:249-254. Branden, Carl and John Tooze, Introduction to Protein Structure,Garland Publishing; New York, 1991. Alberts, Bruce et al., Molecular Biology of the Cell: third edition, Garland Publishing Inc., New York, 1994. Lewin, Benjamin, Genes VI, Oxford University press, New York, 1997. Internet Site--for loop figure- http://www.med.upenn.edu/biocbiop/..._lab/ people/kercher/research2.html
As mentioned above, the NH2 terminal domain of the lac repressor is involved with the DNA binding <>. The NH2 terminal ends can actually be subdivided into two separate functioning domains: the DNA-binding region (residues 1-45) and the hinge region (residues 46-62) <>. The DNA-binding region, also known as the repressor head-piece, contains a helix-turn- helixmotif (HTH). This motif occurs over the first two helixes of the molecule (residues 6-13, 17-24) <>. A small domain with a rich hydrophobic core is formed by the two alpha helixes in the HTH motif (residues 6-25). The hinge region links the DNA-binding region to the core of the protein. This region of the lac repressor allows the DNA-binding region and the core of the protein to move independent of one another. In the presence of DNA, the hinge region has been shown to make specific interactions with the lac operon, orienting the head-piece for DNA-binding. The hinge region is able to do so through the ordering of residues 50-58, which transform from a coil to a helix in the presence of the lac operon <>. In the absence of the lac operator DNA, the residues are "relaxed," allowing the repressor head-piece to dangle freely. The core repressor protein also has two subdomains, an NH2 and COOH terminal end <>. The core of the protein is also known as the inducer binding domain. The two subdomains of this region are very similar and are connected at three sites by the shared amino acid chain. Both subdomains have similar tertiary structures: a six stranded parallel beta sheet, surrounded by four alpha helixes <>. The COOH-terminus of the protein is responsible for the molecule being a tetramere. The tetramere is formed when the alpha helixes of the four monomers associate in an antiparallel fashion <>. Though the molecule is a tetramere, it is easier to think of it as a dimer of dimers. This is due to the skewed, V shape of the molecule. Though all of the monomers of the molecule are oriented in the same direction, the dimers are oriented in different planes, causing the V shape in the protein, with the DNA-binding region at the furthest points of the V. III. Intramolecular Interactions There are four principle clusters of amino acids in the tetramer that comprise the interface between the two monomers of each dimer <> : residues 70-100, 221- 226, 250-260, and 275-285 <>. Point mutations in any of these clusters, excluding 250-260, will result in only the monomeric form of the repressor. The binding between the two dimers is extremely strong due to the number of interactions. Tetramere formation is fairly weak because binding is a result of hydrophobic interactions between leucine residues in the COOH terminal helices of the protein. Both of the binding helices are from different dimers. The leucine heptad repeats of one COOH helix of one monomer are bound to the leucine heptad repeats of the diagnal monomer (residues 338-356) <>. It is clear that these residues are integral to tetramere formation, because mutations at these sites prevent the formation of the tetramere structure, resulting in two unattached dimers. Because of this weak interaction at the COOH end of the molecule, the lac repressor is sometimes considered a "tethered dimer." IV. Intermolecular Interactions A. DNA-Binding Interactions Definite information has yet to be gathered concerning the exact DNA binding interactions between the repressor head-piece <> and the operator sequence of the lac gene. The repressor tetramere is bound to two independent and symmetric operator DNA double helices. The conformation between the two helices in the HTH motif, discussed above, changes upon DNA binding. Residues Asn-25 and His-29 <> have been shown to come in contact with the DNA upon binding. Slipjer et al (1997), have shown that there is a great decrease in the flexibility of the loop joining the two HTH helices due to binding interactions between His-29 of the monomer and Thy-3 of the operator DNA <>. In addition, the residues Gln-18 and Arg-22, have also been shown to have mobility constrict upon repressor binding to DNA, indicating that the two are most likely involved with DNA binding <>. The interactions above occur in the major groove of the DNA. It has also been shown that the leucine residues at position 56 (from each monomer), makes direct contact with the minor groove of the DNA <>. These leucine residues bind close to the center of the operator DNA and act as a lever, prying open the minor groove of the DNA. In addition to being pried open upon repressor binding, the minor groove also decreases in size. The decrease in the size of the minor groove results in the bending of the DNA which occurs when a full repressor tetramere binds to the DNA. The bend in the DNA (total of 60 A radius of curvature) is localized in the center of the operon, where there is more than a 45A turn. DNA bending The DNA fragments bound to the tetramere, are separated by about 25 angstroms. In addition, there are no interaction between the two pieces of DNA. B. Inducer Binding Interactions The inducer molecule, in this case an IPTG molecule, is bound to the lac repressor at the two protein core sub domains <>. The galactoside, IPTG is used as the inducer instead of a lactose molecule, because lactose can be degraded in the cell, while IPTG can not. Due to the IPTG molecule being semi-symmetric, it can interact with the repressor in one of two ways. The repressor has the opportunity to make three or four bonds to the hydroxyl groups of the IPTG inducer molecule. Specifically, interactions through four hydrogen bonds: Asn246, with O2 of IPTG, Arg197 with O3 and O4, and Asp149 with O6. In addition, the Trp220 amino acid is in van der walls contact with an isopropyl group of IPTG <>. There is also a hydrophobic "sugar pocket" with in the core protein, involving the amino acids: Leu73, Ala75, Pro76, Ile79, Trp220, and Phe293 <>. There are a collection of altered lac repressor molecules that can not elicit a release of the DNA upon binding. These molecules are either defective in sugar binding or at transmitting the allosteric signals to the protein binding domain. These mutant molecules are said to display the Is phenotype. Some of the mutations of these Is phenotype molecules are scattered around the molecule, however most are concentrated in five regions: residues 70-80, 90-100, 190-200, 245-250, and 272-277 <>. It is important to realize where these crippling mutations lie because these sites are integral for either inducer binding or allosteric signal transduction. Most of these sites are near the sugar binding site, with the exception of residues 90-100. For this reason, it is hypothesized that these residues are responsible for allosteric signaling. They form the first Beta sheet of the NH2 terminal subdomain of the core domain, located at the dimer interface. C. Allosteric Signal Transduction There are two different structures of the lac repressor molecule corresponding to the induced and repressed states (bound to DNA). It is thought that this change is most likely propagated through the hinge piece discussed above. The change of the NH2 subdomain, as a result of inducer binding, results in a 3.5 angstrom displacement of the alpha carbon of residue 62: this amino acid is the last amino acid of the head-piece, connecting the hinge region to the core protein molecule. The alteration of this residue, results in disruption in the interaction of the hinge helices, a freeing of the HTH bound DNA, and a reduction in the affinity for the operator by the lac repressor molecule. This is similar to the allosteric changes in the oxy form of hemoglobin. Specifically, in the induced state, there are electrostatic alterations between chains in a dimer in the following residues: Lys84 and Glu100, Gln117 and Arg118, and His 74 and Asp 278 <>. The changes in binding between these residues is a result of a 3 angstrom change in the positioning of their alpha carbon. D. Role of The Tetramere If the only allosteric changes in the repressor molecule occure between chains in a dimer, the question arises as to why there is a need for the tetramere structure at all. A single repressor molecule binds to two operators that are between 93 and 401 bp apart. One suggested mechanism is that the repressor dimers bind to separate operator sequences, forcing the DNA to conform to a loop structure. In order for this kind of DNA interaction to occur, there must be a sufficient length between the operator sequences, in addition to certain properties of the DNA. The formation of this loop structure makes DNA polymerase binding even more unfavorable than if only one repressor dimer were bound to the DNA. It is aparent that this mechanism has evolved in cells in order to completely shut off lac operon transcription, when the inducer is bound. This is most likely a result of the extreme importance of metabolism regulation in cells. Another possible mechanism for bending the DNA is to place another DNA binding protein to the DNA loop region causing a sharper bend to the loop. Cap interacts with the operator sequence between the first and second operator in order to induce a 900 bend in the DNA over 30 bp. It has been shown that the CAP repressor combination binds DNA tighter than the repressor molecule alone. This works out well because the production of CAP is increased at times in the cell when energy needs to be conserved, and lactose cleavage needs to be slowed. Chime Index References Lewis, M., Chang G., et al., 1996. "Crystal structure of teh Lactose Operon Repressor and Its Complexes with DNA and Inducer." Science 271:1247-1254. Lamerichs, R.M.J.N., et al., 1989. "H NMR study of a Comples between the lac Repressor Headpiece and a 22 Base Pair Symmetric lac Operator." Biochemistry 28:2985-2991. Friedman, Alan M., et al., 1995. "Crystal Structure of lac Repressor Core Tetramere and Its Implications for DNA looping." Science 268:1721-1727. Slijper, M. et al., 1997. "Backbone and Side Chain Dynamics of lac Repressor Headpiece (1-56) and Its Complex with DNA." Biochemistry 36:249-254. Branden, Carl and John Tooze, Introduction to Protein Structure,Garland Publishing; New York, 1991. Alberts, Bruce et al., Molecular Biology of the Cell: third edition, Garland Publishing Inc., New York, 1994. Lewin, Benjamin, Genes VI, Oxford University press, New York, 1997. Internet Site--for loop figure- http://www.med.upenn.edu/biocbiop/..._lab/ people/kercher/research2.html
The DNA-binding region, also known as the repressor head-piece, contains a helix-turn- helixmotif (HTH). This motif occurs over the first two helixes of the molecule (residues 6-13, 17-24) <>. A small domain with a rich hydrophobic core is formed by the two alpha helixes in the HTH motif (residues 6-25). The hinge region links the DNA-binding region to the core of the protein. This region of the lac repressor allows the DNA-binding region and the core of the protein to move independent of one another. In the presence of DNA, the hinge region has been shown to make specific interactions with the lac operon, orienting the head-piece for DNA-binding. The hinge region is able to do so through the ordering of residues 50-58, which transform from a coil to a helix in the presence of the lac operon <>. In the absence of the lac operator DNA, the residues are "relaxed," allowing the repressor head-piece to dangle freely. The core repressor protein also has two subdomains, an NH2 and COOH terminal end <>. The core of the protein is also known as the inducer binding domain. The two subdomains of this region are very similar and are connected at three sites by the shared amino acid chain. Both subdomains have similar tertiary structures: a six stranded parallel beta sheet, surrounded by four alpha helixes <>. The COOH-terminus of the protein is responsible for the molecule being a tetramere. The tetramere is formed when the alpha helixes of the four monomers associate in an antiparallel fashion <>. Though the molecule is a tetramere, it is easier to think of it as a dimer of dimers. This is due to the skewed, V shape of the molecule. Though all of the monomers of the molecule are oriented in the same direction, the dimers are oriented in different planes, causing the V shape in the protein, with the DNA-binding region at the furthest points of the V. III. Intramolecular Interactions There are four principle clusters of amino acids in the tetramer that comprise the interface between the two monomers of each dimer <> : residues 70-100, 221- 226, 250-260, and 275-285 <>. Point mutations in any of these clusters, excluding 250-260, will result in only the monomeric form of the repressor. The binding between the two dimers is extremely strong due to the number of interactions. Tetramere formation is fairly weak because binding is a result of hydrophobic interactions between leucine residues in the COOH terminal helices of the protein. Both of the binding helices are from different dimers. The leucine heptad repeats of one COOH helix of one monomer are bound to the leucine heptad repeats of the diagnal monomer (residues 338-356) <>. It is clear that these residues are integral to tetramere formation, because mutations at these sites prevent the formation of the tetramere structure, resulting in two unattached dimers. Because of this weak interaction at the COOH end of the molecule, the lac repressor is sometimes considered a "tethered dimer." IV. Intermolecular Interactions A. DNA-Binding Interactions Definite information has yet to be gathered concerning the exact DNA binding interactions between the repressor head-piece <> and the operator sequence of the lac gene. The repressor tetramere is bound to two independent and symmetric operator DNA double helices. The conformation between the two helices in the HTH motif, discussed above, changes upon DNA binding. Residues Asn-25 and His-29 <> have been shown to come in contact with the DNA upon binding. Slipjer et al (1997), have shown that there is a great decrease in the flexibility of the loop joining the two HTH helices due to binding interactions between His-29 of the monomer and Thy-3 of the operator DNA <>. In addition, the residues Gln-18 and Arg-22, have also been shown to have mobility constrict upon repressor binding to DNA, indicating that the two are most likely involved with DNA binding <>. The interactions above occur in the major groove of the DNA. It has also been shown that the leucine residues at position 56 (from each monomer), makes direct contact with the minor groove of the DNA <>. These leucine residues bind close to the center of the operator DNA and act as a lever, prying open the minor groove of the DNA. In addition to being pried open upon repressor binding, the minor groove also decreases in size. The decrease in the size of the minor groove results in the bending of the DNA which occurs when a full repressor tetramere binds to the DNA. The bend in the DNA (total of 60 A radius of curvature) is localized in the center of the operon, where there is more than a 45A turn. DNA bending The DNA fragments bound to the tetramere, are separated by about 25 angstroms. In addition, there are no interaction between the two pieces of DNA. B. Inducer Binding Interactions The inducer molecule, in this case an IPTG molecule, is bound to the lac repressor at the two protein core sub domains <>. The galactoside, IPTG is used as the inducer instead of a lactose molecule, because lactose can be degraded in the cell, while IPTG can not. Due to the IPTG molecule being semi-symmetric, it can interact with the repressor in one of two ways. The repressor has the opportunity to make three or four bonds to the hydroxyl groups of the IPTG inducer molecule. Specifically, interactions through four hydrogen bonds: Asn246, with O2 of IPTG, Arg197 with O3 and O4, and Asp149 with O6. In addition, the Trp220 amino acid is in van der walls contact with an isopropyl group of IPTG <>. There is also a hydrophobic "sugar pocket" with in the core protein, involving the amino acids: Leu73, Ala75, Pro76, Ile79, Trp220, and Phe293 <>. There are a collection of altered lac repressor molecules that can not elicit a release of the DNA upon binding. These molecules are either defective in sugar binding or at transmitting the allosteric signals to the protein binding domain. These mutant molecules are said to display the Is phenotype. Some of the mutations of these Is phenotype molecules are scattered around the molecule, however most are concentrated in five regions: residues 70-80, 90-100, 190-200, 245-250, and 272-277 <>. It is important to realize where these crippling mutations lie because these sites are integral for either inducer binding or allosteric signal transduction. Most of these sites are near the sugar binding site, with the exception of residues 90-100. For this reason, it is hypothesized that these residues are responsible for allosteric signaling. They form the first Beta sheet of the NH2 terminal subdomain of the core domain, located at the dimer interface. C. Allosteric Signal Transduction There are two different structures of the lac repressor molecule corresponding to the induced and repressed states (bound to DNA). It is thought that this change is most likely propagated through the hinge piece discussed above. The change of the NH2 subdomain, as a result of inducer binding, results in a 3.5 angstrom displacement of the alpha carbon of residue 62: this amino acid is the last amino acid of the head-piece, connecting the hinge region to the core protein molecule. The alteration of this residue, results in disruption in the interaction of the hinge helices, a freeing of the HTH bound DNA, and a reduction in the affinity for the operator by the lac repressor molecule. This is similar to the allosteric changes in the oxy form of hemoglobin. Specifically, in the induced state, there are electrostatic alterations between chains in a dimer in the following residues: Lys84 and Glu100, Gln117 and Arg118, and His 74 and Asp 278 <>. The changes in binding between these residues is a result of a 3 angstrom change in the positioning of their alpha carbon. D. Role of The Tetramere If the only allosteric changes in the repressor molecule occure between chains in a dimer, the question arises as to why there is a need for the tetramere structure at all. A single repressor molecule binds to two operators that are between 93 and 401 bp apart. One suggested mechanism is that the repressor dimers bind to separate operator sequences, forcing the DNA to conform to a loop structure. In order for this kind of DNA interaction to occur, there must be a sufficient length between the operator sequences, in addition to certain properties of the DNA. The formation of this loop structure makes DNA polymerase binding even more unfavorable than if only one repressor dimer were bound to the DNA. It is aparent that this mechanism has evolved in cells in order to completely shut off lac operon transcription, when the inducer is bound. This is most likely a result of the extreme importance of metabolism regulation in cells. Another possible mechanism for bending the DNA is to place another DNA binding protein to the DNA loop region causing a sharper bend to the loop. Cap interacts with the operator sequence between the first and second operator in order to induce a 900 bend in the DNA over 30 bp. It has been shown that the CAP repressor combination binds DNA tighter than the repressor molecule alone. This works out well because the production of CAP is increased at times in the cell when energy needs to be conserved, and lactose cleavage needs to be slowed. Chime Index References Lewis, M., Chang G., et al., 1996. "Crystal structure of teh Lactose Operon Repressor and Its Complexes with DNA and Inducer." Science 271:1247-1254. Lamerichs, R.M.J.N., et al., 1989. "H NMR study of a Comples between the lac Repressor Headpiece and a 22 Base Pair Symmetric lac Operator." Biochemistry 28:2985-2991. Friedman, Alan M., et al., 1995. "Crystal Structure of lac Repressor Core Tetramere and Its Implications for DNA looping." Science 268:1721-1727. Slijper, M. et al., 1997. "Backbone and Side Chain Dynamics of lac Repressor Headpiece (1-56) and Its Complex with DNA." Biochemistry 36:249-254. Branden, Carl and John Tooze, Introduction to Protein Structure,Garland Publishing; New York, 1991. Alberts, Bruce et al., Molecular Biology of the Cell: third edition, Garland Publishing Inc., New York, 1994. Lewin, Benjamin, Genes VI, Oxford University press, New York, 1997. Internet Site--for loop figure- http://www.med.upenn.edu/biocbiop/..._lab/ people/kercher/research2.html
The hinge region links the DNA-binding region to the core of the protein. This region of the lac repressor allows the DNA-binding region and the core of the protein to move independent of one another. In the presence of DNA, the hinge region has been shown to make specific interactions with the lac operon, orienting the head-piece for DNA-binding. The hinge region is able to do so through the ordering of residues 50-58, which transform from a coil to a helix in the presence of the lac operon <>. In the absence of the lac operator DNA, the residues are "relaxed," allowing the repressor head-piece to dangle freely. The core repressor protein also has two subdomains, an NH2 and COOH terminal end <>. The core of the protein is also known as the inducer binding domain. The two subdomains of this region are very similar and are connected at three sites by the shared amino acid chain. Both subdomains have similar tertiary structures: a six stranded parallel beta sheet, surrounded by four alpha helixes <>. The COOH-terminus of the protein is responsible for the molecule being a tetramere. The tetramere is formed when the alpha helixes of the four monomers associate in an antiparallel fashion <>. Though the molecule is a tetramere, it is easier to think of it as a dimer of dimers. This is due to the skewed, V shape of the molecule. Though all of the monomers of the molecule are oriented in the same direction, the dimers are oriented in different planes, causing the V shape in the protein, with the DNA-binding region at the furthest points of the V. III. Intramolecular Interactions There are four principle clusters of amino acids in the tetramer that comprise the interface between the two monomers of each dimer <> : residues 70-100, 221- 226, 250-260, and 275-285 <>. Point mutations in any of these clusters, excluding 250-260, will result in only the monomeric form of the repressor. The binding between the two dimers is extremely strong due to the number of interactions. Tetramere formation is fairly weak because binding is a result of hydrophobic interactions between leucine residues in the COOH terminal helices of the protein. Both of the binding helices are from different dimers. The leucine heptad repeats of one COOH helix of one monomer are bound to the leucine heptad repeats of the diagnal monomer (residues 338-356) <>. It is clear that these residues are integral to tetramere formation, because mutations at these sites prevent the formation of the tetramere structure, resulting in two unattached dimers. Because of this weak interaction at the COOH end of the molecule, the lac repressor is sometimes considered a "tethered dimer." IV. Intermolecular Interactions A. DNA-Binding Interactions Definite information has yet to be gathered concerning the exact DNA binding interactions between the repressor head-piece <> and the operator sequence of the lac gene. The repressor tetramere is bound to two independent and symmetric operator DNA double helices. The conformation between the two helices in the HTH motif, discussed above, changes upon DNA binding. Residues Asn-25 and His-29 <> have been shown to come in contact with the DNA upon binding. Slipjer et al (1997), have shown that there is a great decrease in the flexibility of the loop joining the two HTH helices due to binding interactions between His-29 of the monomer and Thy-3 of the operator DNA <>. In addition, the residues Gln-18 and Arg-22, have also been shown to have mobility constrict upon repressor binding to DNA, indicating that the two are most likely involved with DNA binding <>. The interactions above occur in the major groove of the DNA. It has also been shown that the leucine residues at position 56 (from each monomer), makes direct contact with the minor groove of the DNA <>. These leucine residues bind close to the center of the operator DNA and act as a lever, prying open the minor groove of the DNA. In addition to being pried open upon repressor binding, the minor groove also decreases in size. The decrease in the size of the minor groove results in the bending of the DNA which occurs when a full repressor tetramere binds to the DNA. The bend in the DNA (total of 60 A radius of curvature) is localized in the center of the operon, where there is more than a 45A turn. DNA bending The DNA fragments bound to the tetramere, are separated by about 25 angstroms. In addition, there are no interaction between the two pieces of DNA. B. Inducer Binding Interactions The inducer molecule, in this case an IPTG molecule, is bound to the lac repressor at the two protein core sub domains <>. The galactoside, IPTG is used as the inducer instead of a lactose molecule, because lactose can be degraded in the cell, while IPTG can not. Due to the IPTG molecule being semi-symmetric, it can interact with the repressor in one of two ways. The repressor has the opportunity to make three or four bonds to the hydroxyl groups of the IPTG inducer molecule. Specifically, interactions through four hydrogen bonds: Asn246, with O2 of IPTG, Arg197 with O3 and O4, and Asp149 with O6. In addition, the Trp220 amino acid is in van der walls contact with an isopropyl group of IPTG <>. There is also a hydrophobic "sugar pocket" with in the core protein, involving the amino acids: Leu73, Ala75, Pro76, Ile79, Trp220, and Phe293 <>. There are a collection of altered lac repressor molecules that can not elicit a release of the DNA upon binding. These molecules are either defective in sugar binding or at transmitting the allosteric signals to the protein binding domain. These mutant molecules are said to display the Is phenotype. Some of the mutations of these Is phenotype molecules are scattered around the molecule, however most are concentrated in five regions: residues 70-80, 90-100, 190-200, 245-250, and 272-277 <>. It is important to realize where these crippling mutations lie because these sites are integral for either inducer binding or allosteric signal transduction. Most of these sites are near the sugar binding site, with the exception of residues 90-100. For this reason, it is hypothesized that these residues are responsible for allosteric signaling. They form the first Beta sheet of the NH2 terminal subdomain of the core domain, located at the dimer interface. C. Allosteric Signal Transduction There are two different structures of the lac repressor molecule corresponding to the induced and repressed states (bound to DNA). It is thought that this change is most likely propagated through the hinge piece discussed above. The change of the NH2 subdomain, as a result of inducer binding, results in a 3.5 angstrom displacement of the alpha carbon of residue 62: this amino acid is the last amino acid of the head-piece, connecting the hinge region to the core protein molecule. The alteration of this residue, results in disruption in the interaction of the hinge helices, a freeing of the HTH bound DNA, and a reduction in the affinity for the operator by the lac repressor molecule. This is similar to the allosteric changes in the oxy form of hemoglobin. Specifically, in the induced state, there are electrostatic alterations between chains in a dimer in the following residues: Lys84 and Glu100, Gln117 and Arg118, and His 74 and Asp 278 <>. The changes in binding between these residues is a result of a 3 angstrom change in the positioning of their alpha carbon. D. Role of The Tetramere If the only allosteric changes in the repressor molecule occure between chains in a dimer, the question arises as to why there is a need for the tetramere structure at all. A single repressor molecule binds to two operators that are between 93 and 401 bp apart. One suggested mechanism is that the repressor dimers bind to separate operator sequences, forcing the DNA to conform to a loop structure. In order for this kind of DNA interaction to occur, there must be a sufficient length between the operator sequences, in addition to certain properties of the DNA. The formation of this loop structure makes DNA polymerase binding even more unfavorable than if only one repressor dimer were bound to the DNA. It is aparent that this mechanism has evolved in cells in order to completely shut off lac operon transcription, when the inducer is bound. This is most likely a result of the extreme importance of metabolism regulation in cells. Another possible mechanism for bending the DNA is to place another DNA binding protein to the DNA loop region causing a sharper bend to the loop. Cap interacts with the operator sequence between the first and second operator in order to induce a 900 bend in the DNA over 30 bp. It has been shown that the CAP repressor combination binds DNA tighter than the repressor molecule alone. This works out well because the production of CAP is increased at times in the cell when energy needs to be conserved, and lactose cleavage needs to be slowed. Chime Index References Lewis, M., Chang G., et al., 1996. "Crystal structure of teh Lactose Operon Repressor and Its Complexes with DNA and Inducer." Science 271:1247-1254. Lamerichs, R.M.J.N., et al., 1989. "H NMR study of a Comples between the lac Repressor Headpiece and a 22 Base Pair Symmetric lac Operator." Biochemistry 28:2985-2991. Friedman, Alan M., et al., 1995. "Crystal Structure of lac Repressor Core Tetramere and Its Implications for DNA looping." Science 268:1721-1727. Slijper, M. et al., 1997. "Backbone and Side Chain Dynamics of lac Repressor Headpiece (1-56) and Its Complex with DNA." Biochemistry 36:249-254. Branden, Carl and John Tooze, Introduction to Protein Structure,Garland Publishing; New York, 1991. Alberts, Bruce et al., Molecular Biology of the Cell: third edition, Garland Publishing Inc., New York, 1994. Lewin, Benjamin, Genes VI, Oxford University press, New York, 1997. Internet Site--for loop figure- http://www.med.upenn.edu/biocbiop/..._lab/ people/kercher/research2.html
The core repressor protein also has two subdomains, an NH2 and COOH terminal end <>. The core of the protein is also known as the inducer binding domain. The two subdomains of this region are very similar and are connected at three sites by the shared amino acid chain. Both subdomains have similar tertiary structures: a six stranded parallel beta sheet, surrounded by four alpha helixes <>. The COOH-terminus of the protein is responsible for the molecule being a tetramere. The tetramere is formed when the alpha helixes of the four monomers associate in an antiparallel fashion <>. Though the molecule is a tetramere, it is easier to think of it as a dimer of dimers. This is due to the skewed, V shape of the molecule. Though all of the monomers of the molecule are oriented in the same direction, the dimers are oriented in different planes, causing the V shape in the protein, with the DNA-binding region at the furthest points of the V. III. Intramolecular Interactions There are four principle clusters of amino acids in the tetramer that comprise the interface between the two monomers of each dimer <> : residues 70-100, 221- 226, 250-260, and 275-285 <>. Point mutations in any of these clusters, excluding 250-260, will result in only the monomeric form of the repressor. The binding between the two dimers is extremely strong due to the number of interactions. Tetramere formation is fairly weak because binding is a result of hydrophobic interactions between leucine residues in the COOH terminal helices of the protein. Both of the binding helices are from different dimers. The leucine heptad repeats of one COOH helix of one monomer are bound to the leucine heptad repeats of the diagnal monomer (residues 338-356) <>. It is clear that these residues are integral to tetramere formation, because mutations at these sites prevent the formation of the tetramere structure, resulting in two unattached dimers. Because of this weak interaction at the COOH end of the molecule, the lac repressor is sometimes considered a "tethered dimer." IV. Intermolecular Interactions A. DNA-Binding Interactions Definite information has yet to be gathered concerning the exact DNA binding interactions between the repressor head-piece <> and the operator sequence of the lac gene. The repressor tetramere is bound to two independent and symmetric operator DNA double helices. The conformation between the two helices in the HTH motif, discussed above, changes upon DNA binding. Residues Asn-25 and His-29 <> have been shown to come in contact with the DNA upon binding. Slipjer et al (1997), have shown that there is a great decrease in the flexibility of the loop joining the two HTH helices due to binding interactions between His-29 of the monomer and Thy-3 of the operator DNA <>. In addition, the residues Gln-18 and Arg-22, have also been shown to have mobility constrict upon repressor binding to DNA, indicating that the two are most likely involved with DNA binding <>. The interactions above occur in the major groove of the DNA. It has also been shown that the leucine residues at position 56 (from each monomer), makes direct contact with the minor groove of the DNA <>. These leucine residues bind close to the center of the operator DNA and act as a lever, prying open the minor groove of the DNA. In addition to being pried open upon repressor binding, the minor groove also decreases in size. The decrease in the size of the minor groove results in the bending of the DNA which occurs when a full repressor tetramere binds to the DNA. The bend in the DNA (total of 60 A radius of curvature) is localized in the center of the operon, where there is more than a 45A turn. DNA bending The DNA fragments bound to the tetramere, are separated by about 25 angstroms. In addition, there are no interaction between the two pieces of DNA. B. Inducer Binding Interactions The inducer molecule, in this case an IPTG molecule, is bound to the lac repressor at the two protein core sub domains <>. The galactoside, IPTG is used as the inducer instead of a lactose molecule, because lactose can be degraded in the cell, while IPTG can not. Due to the IPTG molecule being semi-symmetric, it can interact with the repressor in one of two ways. The repressor has the opportunity to make three or four bonds to the hydroxyl groups of the IPTG inducer molecule. Specifically, interactions through four hydrogen bonds: Asn246, with O2 of IPTG, Arg197 with O3 and O4, and Asp149 with O6. In addition, the Trp220 amino acid is in van der walls contact with an isopropyl group of IPTG <>. There is also a hydrophobic "sugar pocket" with in the core protein, involving the amino acids: Leu73, Ala75, Pro76, Ile79, Trp220, and Phe293 <>. There are a collection of altered lac repressor molecules that can not elicit a release of the DNA upon binding. These molecules are either defective in sugar binding or at transmitting the allosteric signals to the protein binding domain. These mutant molecules are said to display the Is phenotype. Some of the mutations of these Is phenotype molecules are scattered around the molecule, however most are concentrated in five regions: residues 70-80, 90-100, 190-200, 245-250, and 272-277 <>. It is important to realize where these crippling mutations lie because these sites are integral for either inducer binding or allosteric signal transduction. Most of these sites are near the sugar binding site, with the exception of residues 90-100. For this reason, it is hypothesized that these residues are responsible for allosteric signaling. They form the first Beta sheet of the NH2 terminal subdomain of the core domain, located at the dimer interface. C. Allosteric Signal Transduction There are two different structures of the lac repressor molecule corresponding to the induced and repressed states (bound to DNA). It is thought that this change is most likely propagated through the hinge piece discussed above. The change of the NH2 subdomain, as a result of inducer binding, results in a 3.5 angstrom displacement of the alpha carbon of residue 62: this amino acid is the last amino acid of the head-piece, connecting the hinge region to the core protein molecule. The alteration of this residue, results in disruption in the interaction of the hinge helices, a freeing of the HTH bound DNA, and a reduction in the affinity for the operator by the lac repressor molecule. This is similar to the allosteric changes in the oxy form of hemoglobin. Specifically, in the induced state, there are electrostatic alterations between chains in a dimer in the following residues: Lys84 and Glu100, Gln117 and Arg118, and His 74 and Asp 278 <>. The changes in binding between these residues is a result of a 3 angstrom change in the positioning of their alpha carbon. D. Role of The Tetramere If the only allosteric changes in the repressor molecule occure between chains in a dimer, the question arises as to why there is a need for the tetramere structure at all. A single repressor molecule binds to two operators that are between 93 and 401 bp apart. One suggested mechanism is that the repressor dimers bind to separate operator sequences, forcing the DNA to conform to a loop structure. In order for this kind of DNA interaction to occur, there must be a sufficient length between the operator sequences, in addition to certain properties of the DNA. The formation of this loop structure makes DNA polymerase binding even more unfavorable than if only one repressor dimer were bound to the DNA. It is aparent that this mechanism has evolved in cells in order to completely shut off lac operon transcription, when the inducer is bound. This is most likely a result of the extreme importance of metabolism regulation in cells. Another possible mechanism for bending the DNA is to place another DNA binding protein to the DNA loop region causing a sharper bend to the loop. Cap interacts with the operator sequence between the first and second operator in order to induce a 900 bend in the DNA over 30 bp. It has been shown that the CAP repressor combination binds DNA tighter than the repressor molecule alone. This works out well because the production of CAP is increased at times in the cell when energy needs to be conserved, and lactose cleavage needs to be slowed. Chime Index References Lewis, M., Chang G., et al., 1996. "Crystal structure of teh Lactose Operon Repressor and Its Complexes with DNA and Inducer." Science 271:1247-1254. Lamerichs, R.M.J.N., et al., 1989. "H NMR study of a Comples between the lac Repressor Headpiece and a 22 Base Pair Symmetric lac Operator." Biochemistry 28:2985-2991. Friedman, Alan M., et al., 1995. "Crystal Structure of lac Repressor Core Tetramere and Its Implications for DNA looping." Science 268:1721-1727. Slijper, M. et al., 1997. "Backbone and Side Chain Dynamics of lac Repressor Headpiece (1-56) and Its Complex with DNA." Biochemistry 36:249-254. Branden, Carl and John Tooze, Introduction to Protein Structure,Garland Publishing; New York, 1991. Alberts, Bruce et al., Molecular Biology of the Cell: third edition, Garland Publishing Inc., New York, 1994. Lewin, Benjamin, Genes VI, Oxford University press, New York, 1997. Internet Site--for loop figure- http://www.med.upenn.edu/biocbiop/..._lab/ people/kercher/research2.html
The COOH-terminus of the protein is responsible for the molecule being a tetramere. The tetramere is formed when the alpha helixes of the four monomers associate in an antiparallel fashion <>. Though the molecule is a tetramere, it is easier to think of it as a dimer of dimers. This is due to the skewed, V shape of the molecule. Though all of the monomers of the molecule are oriented in the same direction, the dimers are oriented in different planes, causing the V shape in the protein, with the DNA-binding region at the furthest points of the V. III. Intramolecular Interactions There are four principle clusters of amino acids in the tetramer that comprise the interface between the two monomers of each dimer <> : residues 70-100, 221- 226, 250-260, and 275-285 <>. Point mutations in any of these clusters, excluding 250-260, will result in only the monomeric form of the repressor. The binding between the two dimers is extremely strong due to the number of interactions. Tetramere formation is fairly weak because binding is a result of hydrophobic interactions between leucine residues in the COOH terminal helices of the protein. Both of the binding helices are from different dimers. The leucine heptad repeats of one COOH helix of one monomer are bound to the leucine heptad repeats of the diagnal monomer (residues 338-356) <>. It is clear that these residues are integral to tetramere formation, because mutations at these sites prevent the formation of the tetramere structure, resulting in two unattached dimers. Because of this weak interaction at the COOH end of the molecule, the lac repressor is sometimes considered a "tethered dimer." IV. Intermolecular Interactions A. DNA-Binding Interactions Definite information has yet to be gathered concerning the exact DNA binding interactions between the repressor head-piece <> and the operator sequence of the lac gene. The repressor tetramere is bound to two independent and symmetric operator DNA double helices. The conformation between the two helices in the HTH motif, discussed above, changes upon DNA binding. Residues Asn-25 and His-29 <> have been shown to come in contact with the DNA upon binding. Slipjer et al (1997), have shown that there is a great decrease in the flexibility of the loop joining the two HTH helices due to binding interactions between His-29 of the monomer and Thy-3 of the operator DNA <>. In addition, the residues Gln-18 and Arg-22, have also been shown to have mobility constrict upon repressor binding to DNA, indicating that the two are most likely involved with DNA binding <>. The interactions above occur in the major groove of the DNA. It has also been shown that the leucine residues at position 56 (from each monomer), makes direct contact with the minor groove of the DNA <>. These leucine residues bind close to the center of the operator DNA and act as a lever, prying open the minor groove of the DNA. In addition to being pried open upon repressor binding, the minor groove also decreases in size. The decrease in the size of the minor groove results in the bending of the DNA which occurs when a full repressor tetramere binds to the DNA. The bend in the DNA (total of 60 A radius of curvature) is localized in the center of the operon, where there is more than a 45A turn. DNA bending The DNA fragments bound to the tetramere, are separated by about 25 angstroms. In addition, there are no interaction between the two pieces of DNA. B. Inducer Binding Interactions The inducer molecule, in this case an IPTG molecule, is bound to the lac repressor at the two protein core sub domains <>. The galactoside, IPTG is used as the inducer instead of a lactose molecule, because lactose can be degraded in the cell, while IPTG can not. Due to the IPTG molecule being semi-symmetric, it can interact with the repressor in one of two ways. The repressor has the opportunity to make three or four bonds to the hydroxyl groups of the IPTG inducer molecule. Specifically, interactions through four hydrogen bonds: Asn246, with O2 of IPTG, Arg197 with O3 and O4, and Asp149 with O6. In addition, the Trp220 amino acid is in van der walls contact with an isopropyl group of IPTG <>. There is also a hydrophobic "sugar pocket" with in the core protein, involving the amino acids: Leu73, Ala75, Pro76, Ile79, Trp220, and Phe293 <>. There are a collection of altered lac repressor molecules that can not elicit a release of the DNA upon binding. These molecules are either defective in sugar binding or at transmitting the allosteric signals to the protein binding domain. These mutant molecules are said to display the Is phenotype. Some of the mutations of these Is phenotype molecules are scattered around the molecule, however most are concentrated in five regions: residues 70-80, 90-100, 190-200, 245-250, and 272-277 <>. It is important to realize where these crippling mutations lie because these sites are integral for either inducer binding or allosteric signal transduction. Most of these sites are near the sugar binding site, with the exception of residues 90-100. For this reason, it is hypothesized that these residues are responsible for allosteric signaling. They form the first Beta sheet of the NH2 terminal subdomain of the core domain, located at the dimer interface. C. Allosteric Signal Transduction There are two different structures of the lac repressor molecule corresponding to the induced and repressed states (bound to DNA). It is thought that this change is most likely propagated through the hinge piece discussed above. The change of the NH2 subdomain, as a result of inducer binding, results in a 3.5 angstrom displacement of the alpha carbon of residue 62: this amino acid is the last amino acid of the head-piece, connecting the hinge region to the core protein molecule. The alteration of this residue, results in disruption in the interaction of the hinge helices, a freeing of the HTH bound DNA, and a reduction in the affinity for the operator by the lac repressor molecule. This is similar to the allosteric changes in the oxy form of hemoglobin. Specifically, in the induced state, there are electrostatic alterations between chains in a dimer in the following residues: Lys84 and Glu100, Gln117 and Arg118, and His 74 and Asp 278 <>. The changes in binding between these residues is a result of a 3 angstrom change in the positioning of their alpha carbon. D. Role of The Tetramere If the only allosteric changes in the repressor molecule occure between chains in a dimer, the question arises as to why there is a need for the tetramere structure at all. A single repressor molecule binds to two operators that are between 93 and 401 bp apart. One suggested mechanism is that the repressor dimers bind to separate operator sequences, forcing the DNA to conform to a loop structure. In order for this kind of DNA interaction to occur, there must be a sufficient length between the operator sequences, in addition to certain properties of the DNA. The formation of this loop structure makes DNA polymerase binding even more unfavorable than if only one repressor dimer were bound to the DNA. It is aparent that this mechanism has evolved in cells in order to completely shut off lac operon transcription, when the inducer is bound. This is most likely a result of the extreme importance of metabolism regulation in cells. Another possible mechanism for bending the DNA is to place another DNA binding protein to the DNA loop region causing a sharper bend to the loop. Cap interacts with the operator sequence between the first and second operator in order to induce a 900 bend in the DNA over 30 bp. It has been shown that the CAP repressor combination binds DNA tighter than the repressor molecule alone. This works out well because the production of CAP is increased at times in the cell when energy needs to be conserved, and lactose cleavage needs to be slowed. Chime Index References Lewis, M., Chang G., et al., 1996. "Crystal structure of teh Lactose Operon Repressor and Its Complexes with DNA and Inducer." Science 271:1247-1254. Lamerichs, R.M.J.N., et al., 1989. "H NMR study of a Comples between the lac Repressor Headpiece and a 22 Base Pair Symmetric lac Operator." Biochemistry 28:2985-2991. Friedman, Alan M., et al., 1995. "Crystal Structure of lac Repressor Core Tetramere and Its Implications for DNA looping." Science 268:1721-1727. Slijper, M. et al., 1997. "Backbone and Side Chain Dynamics of lac Repressor Headpiece (1-56) and Its Complex with DNA." Biochemistry 36:249-254. Branden, Carl and John Tooze, Introduction to Protein Structure,Garland Publishing; New York, 1991. Alberts, Bruce et al., Molecular Biology of the Cell: third edition, Garland Publishing Inc., New York, 1994. Lewin, Benjamin, Genes VI, Oxford University press, New York, 1997. Internet Site--for loop figure- http://www.med.upenn.edu/biocbiop/..._lab/ people/kercher/research2.html
Tetramere formation is fairly weak because binding is a result of hydrophobic interactions between leucine residues in the COOH terminal helices of the protein. Both of the binding helices are from different dimers. The leucine heptad repeats of one COOH helix of one monomer are bound to the leucine heptad repeats of the diagnal monomer (residues 338-356) <>. It is clear that these residues are integral to tetramere formation, because mutations at these sites prevent the formation of the tetramere structure, resulting in two unattached dimers. Because of this weak interaction at the COOH end of the molecule, the lac repressor is sometimes considered a "tethered dimer." IV. Intermolecular Interactions A. DNA-Binding Interactions Definite information has yet to be gathered concerning the exact DNA binding interactions between the repressor head-piece <> and the operator sequence of the lac gene. The repressor tetramere is bound to two independent and symmetric operator DNA double helices. The conformation between the two helices in the HTH motif, discussed above, changes upon DNA binding. Residues Asn-25 and His-29 <> have been shown to come in contact with the DNA upon binding. Slipjer et al (1997), have shown that there is a great decrease in the flexibility of the loop joining the two HTH helices due to binding interactions between His-29 of the monomer and Thy-3 of the operator DNA <>. In addition, the residues Gln-18 and Arg-22, have also been shown to have mobility constrict upon repressor binding to DNA, indicating that the two are most likely involved with DNA binding <>. The interactions above occur in the major groove of the DNA. It has also been shown that the leucine residues at position 56 (from each monomer), makes direct contact with the minor groove of the DNA <>. These leucine residues bind close to the center of the operator DNA and act as a lever, prying open the minor groove of the DNA. In addition to being pried open upon repressor binding, the minor groove also decreases in size. The decrease in the size of the minor groove results in the bending of the DNA which occurs when a full repressor tetramere binds to the DNA. The bend in the DNA (total of 60 A radius of curvature) is localized in the center of the operon, where there is more than a 45A turn. DNA bending The DNA fragments bound to the tetramere, are separated by about 25 angstroms. In addition, there are no interaction between the two pieces of DNA. B. Inducer Binding Interactions The inducer molecule, in this case an IPTG molecule, is bound to the lac repressor at the two protein core sub domains <>. The galactoside, IPTG is used as the inducer instead of a lactose molecule, because lactose can be degraded in the cell, while IPTG can not. Due to the IPTG molecule being semi-symmetric, it can interact with the repressor in one of two ways. The repressor has the opportunity to make three or four bonds to the hydroxyl groups of the IPTG inducer molecule. Specifically, interactions through four hydrogen bonds: Asn246, with O2 of IPTG, Arg197 with O3 and O4, and Asp149 with O6. In addition, the Trp220 amino acid is in van der walls contact with an isopropyl group of IPTG <>. There is also a hydrophobic "sugar pocket" with in the core protein, involving the amino acids: Leu73, Ala75, Pro76, Ile79, Trp220, and Phe293 <>. There are a collection of altered lac repressor molecules that can not elicit a release of the DNA upon binding. These molecules are either defective in sugar binding or at transmitting the allosteric signals to the protein binding domain. These mutant molecules are said to display the Is phenotype. Some of the mutations of these Is phenotype molecules are scattered around the molecule, however most are concentrated in five regions: residues 70-80, 90-100, 190-200, 245-250, and 272-277 <>. It is important to realize where these crippling mutations lie because these sites are integral for either inducer binding or allosteric signal transduction. Most of these sites are near the sugar binding site, with the exception of residues 90-100. For this reason, it is hypothesized that these residues are responsible for allosteric signaling. They form the first Beta sheet of the NH2 terminal subdomain of the core domain, located at the dimer interface. C. Allosteric Signal Transduction There are two different structures of the lac repressor molecule corresponding to the induced and repressed states (bound to DNA). It is thought that this change is most likely propagated through the hinge piece discussed above. The change of the NH2 subdomain, as a result of inducer binding, results in a 3.5 angstrom displacement of the alpha carbon of residue 62: this amino acid is the last amino acid of the head-piece, connecting the hinge region to the core protein molecule. The alteration of this residue, results in disruption in the interaction of the hinge helices, a freeing of the HTH bound DNA, and a reduction in the affinity for the operator by the lac repressor molecule. This is similar to the allosteric changes in the oxy form of hemoglobin. Specifically, in the induced state, there are electrostatic alterations between chains in a dimer in the following residues: Lys84 and Glu100, Gln117 and Arg118, and His 74 and Asp 278 <>. The changes in binding between these residues is a result of a 3 angstrom change in the positioning of their alpha carbon. D. Role of The Tetramere If the only allosteric changes in the repressor molecule occure between chains in a dimer, the question arises as to why there is a need for the tetramere structure at all. A single repressor molecule binds to two operators that are between 93 and 401 bp apart. One suggested mechanism is that the repressor dimers bind to separate operator sequences, forcing the DNA to conform to a loop structure. In order for this kind of DNA interaction to occur, there must be a sufficient length between the operator sequences, in addition to certain properties of the DNA. The formation of this loop structure makes DNA polymerase binding even more unfavorable than if only one repressor dimer were bound to the DNA. It is aparent that this mechanism has evolved in cells in order to completely shut off lac operon transcription, when the inducer is bound. This is most likely a result of the extreme importance of metabolism regulation in cells. Another possible mechanism for bending the DNA is to place another DNA binding protein to the DNA loop region causing a sharper bend to the loop. Cap interacts with the operator sequence between the first and second operator in order to induce a 900 bend in the DNA over 30 bp. It has been shown that the CAP repressor combination binds DNA tighter than the repressor molecule alone. This works out well because the production of CAP is increased at times in the cell when energy needs to be conserved, and lactose cleavage needs to be slowed. Chime Index References Lewis, M., Chang G., et al., 1996. "Crystal structure of teh Lactose Operon Repressor and Its Complexes with DNA and Inducer." Science 271:1247-1254. Lamerichs, R.M.J.N., et al., 1989. "H NMR study of a Comples between the lac Repressor Headpiece and a 22 Base Pair Symmetric lac Operator." Biochemistry 28:2985-2991. Friedman, Alan M., et al., 1995. "Crystal Structure of lac Repressor Core Tetramere and Its Implications for DNA looping." Science 268:1721-1727. Slijper, M. et al., 1997. "Backbone and Side Chain Dynamics of lac Repressor Headpiece (1-56) and Its Complex with DNA." Biochemistry 36:249-254. Branden, Carl and John Tooze, Introduction to Protein Structure,Garland Publishing; New York, 1991. Alberts, Bruce et al., Molecular Biology of the Cell: third edition, Garland Publishing Inc., New York, 1994. Lewin, Benjamin, Genes VI, Oxford University press, New York, 1997. Internet Site--for loop figure- http://www.med.upenn.edu/biocbiop/..._lab/ people/kercher/research2.html
Definite information has yet to be gathered concerning the exact DNA binding interactions between the repressor head-piece <> and the operator sequence of the lac gene. The repressor tetramere is bound to two independent and symmetric operator DNA double helices. The conformation between the two helices in the HTH motif, discussed above, changes upon DNA binding. Residues Asn-25 and His-29 <> have been shown to come in contact with the DNA upon binding. Slipjer et al (1997), have shown that there is a great decrease in the flexibility of the loop joining the two HTH helices due to binding interactions between His-29 of the monomer and Thy-3 of the operator DNA <>. In addition, the residues Gln-18 and Arg-22, have also been shown to have mobility constrict upon repressor binding to DNA, indicating that the two are most likely involved with DNA binding <>. The interactions above occur in the major groove of the DNA. It has also been shown that the leucine residues at position 56 (from each monomer), makes direct contact with the minor groove of the DNA <>. These leucine residues bind close to the center of the operator DNA and act as a lever, prying open the minor groove of the DNA. In addition to being pried open upon repressor binding, the minor groove also decreases in size. The decrease in the size of the minor groove results in the bending of the DNA which occurs when a full repressor tetramere binds to the DNA. The bend in the DNA (total of 60 A radius of curvature) is localized in the center of the operon, where there is more than a 45A turn. DNA bending The DNA fragments bound to the tetramere, are separated by about 25 angstroms. In addition, there are no interaction between the two pieces of DNA. B. Inducer Binding Interactions The inducer molecule, in this case an IPTG molecule, is bound to the lac repressor at the two protein core sub domains <>. The galactoside, IPTG is used as the inducer instead of a lactose molecule, because lactose can be degraded in the cell, while IPTG can not. Due to the IPTG molecule being semi-symmetric, it can interact with the repressor in one of two ways. The repressor has the opportunity to make three or four bonds to the hydroxyl groups of the IPTG inducer molecule. Specifically, interactions through four hydrogen bonds: Asn246, with O2 of IPTG, Arg197 with O3 and O4, and Asp149 with O6. In addition, the Trp220 amino acid is in van der walls contact with an isopropyl group of IPTG <>. There is also a hydrophobic "sugar pocket" with in the core protein, involving the amino acids: Leu73, Ala75, Pro76, Ile79, Trp220, and Phe293 <>. There are a collection of altered lac repressor molecules that can not elicit a release of the DNA upon binding. These molecules are either defective in sugar binding or at transmitting the allosteric signals to the protein binding domain. These mutant molecules are said to display the Is phenotype. Some of the mutations of these Is phenotype molecules are scattered around the molecule, however most are concentrated in five regions: residues 70-80, 90-100, 190-200, 245-250, and 272-277 <>. It is important to realize where these crippling mutations lie because these sites are integral for either inducer binding or allosteric signal transduction. Most of these sites are near the sugar binding site, with the exception of residues 90-100. For this reason, it is hypothesized that these residues are responsible for allosteric signaling. They form the first Beta sheet of the NH2 terminal subdomain of the core domain, located at the dimer interface. C. Allosteric Signal Transduction There are two different structures of the lac repressor molecule corresponding to the induced and repressed states (bound to DNA). It is thought that this change is most likely propagated through the hinge piece discussed above. The change of the NH2 subdomain, as a result of inducer binding, results in a 3.5 angstrom displacement of the alpha carbon of residue 62: this amino acid is the last amino acid of the head-piece, connecting the hinge region to the core protein molecule. The alteration of this residue, results in disruption in the interaction of the hinge helices, a freeing of the HTH bound DNA, and a reduction in the affinity for the operator by the lac repressor molecule. This is similar to the allosteric changes in the oxy form of hemoglobin. Specifically, in the induced state, there are electrostatic alterations between chains in a dimer in the following residues: Lys84 and Glu100, Gln117 and Arg118, and His 74 and Asp 278 <>. The changes in binding between these residues is a result of a 3 angstrom change in the positioning of their alpha carbon. D. Role of The Tetramere If the only allosteric changes in the repressor molecule occure between chains in a dimer, the question arises as to why there is a need for the tetramere structure at all. A single repressor molecule binds to two operators that are between 93 and 401 bp apart. One suggested mechanism is that the repressor dimers bind to separate operator sequences, forcing the DNA to conform to a loop structure. In order for this kind of DNA interaction to occur, there must be a sufficient length between the operator sequences, in addition to certain properties of the DNA. The formation of this loop structure makes DNA polymerase binding even more unfavorable than if only one repressor dimer were bound to the DNA. It is aparent that this mechanism has evolved in cells in order to completely shut off lac operon transcription, when the inducer is bound. This is most likely a result of the extreme importance of metabolism regulation in cells. Another possible mechanism for bending the DNA is to place another DNA binding protein to the DNA loop region causing a sharper bend to the loop. Cap interacts with the operator sequence between the first and second operator in order to induce a 900 bend in the DNA over 30 bp. It has been shown that the CAP repressor combination binds DNA tighter than the repressor molecule alone. This works out well because the production of CAP is increased at times in the cell when energy needs to be conserved, and lactose cleavage needs to be slowed. Chime Index References Lewis, M., Chang G., et al., 1996. "Crystal structure of teh Lactose Operon Repressor and Its Complexes with DNA and Inducer." Science 271:1247-1254. Lamerichs, R.M.J.N., et al., 1989. "H NMR study of a Comples between the lac Repressor Headpiece and a 22 Base Pair Symmetric lac Operator." Biochemistry 28:2985-2991. Friedman, Alan M., et al., 1995. "Crystal Structure of lac Repressor Core Tetramere and Its Implications for DNA looping." Science 268:1721-1727. Slijper, M. et al., 1997. "Backbone and Side Chain Dynamics of lac Repressor Headpiece (1-56) and Its Complex with DNA." Biochemistry 36:249-254. Branden, Carl and John Tooze, Introduction to Protein Structure,Garland Publishing; New York, 1991. Alberts, Bruce et al., Molecular Biology of the Cell: third edition, Garland Publishing Inc., New York, 1994. Lewin, Benjamin, Genes VI, Oxford University press, New York, 1997. Internet Site--for loop figure- http://www.med.upenn.edu/biocbiop/..._lab/ people/kercher/research2.html
The interactions above occur in the major groove of the DNA. It has also been shown that the leucine residues at position 56 (from each monomer), makes direct contact with the minor groove of the DNA <>. These leucine residues bind close to the center of the operator DNA and act as a lever, prying open the minor groove of the DNA. In addition to being pried open upon repressor binding, the minor groove also decreases in size. The decrease in the size of the minor groove results in the bending of the DNA which occurs when a full repressor tetramere binds to the DNA. The bend in the DNA (total of 60 A radius of curvature) is localized in the center of the operon, where there is more than a 45A turn. DNA bending The DNA fragments bound to the tetramere, are separated by about 25 angstroms. In addition, there are no interaction between the two pieces of DNA. B. Inducer Binding Interactions The inducer molecule, in this case an IPTG molecule, is bound to the lac repressor at the two protein core sub domains <>. The galactoside, IPTG is used as the inducer instead of a lactose molecule, because lactose can be degraded in the cell, while IPTG can not. Due to the IPTG molecule being semi-symmetric, it can interact with the repressor in one of two ways. The repressor has the opportunity to make three or four bonds to the hydroxyl groups of the IPTG inducer molecule. Specifically, interactions through four hydrogen bonds: Asn246, with O2 of IPTG, Arg197 with O3 and O4, and Asp149 with O6. In addition, the Trp220 amino acid is in van der walls contact with an isopropyl group of IPTG <>. There is also a hydrophobic "sugar pocket" with in the core protein, involving the amino acids: Leu73, Ala75, Pro76, Ile79, Trp220, and Phe293 <>. There are a collection of altered lac repressor molecules that can not elicit a release of the DNA upon binding. These molecules are either defective in sugar binding or at transmitting the allosteric signals to the protein binding domain. These mutant molecules are said to display the Is phenotype. Some of the mutations of these Is phenotype molecules are scattered around the molecule, however most are concentrated in five regions: residues 70-80, 90-100, 190-200, 245-250, and 272-277 <>. It is important to realize where these crippling mutations lie because these sites are integral for either inducer binding or allosteric signal transduction. Most of these sites are near the sugar binding site, with the exception of residues 90-100. For this reason, it is hypothesized that these residues are responsible for allosteric signaling. They form the first Beta sheet of the NH2 terminal subdomain of the core domain, located at the dimer interface. C. Allosteric Signal Transduction There are two different structures of the lac repressor molecule corresponding to the induced and repressed states (bound to DNA). It is thought that this change is most likely propagated through the hinge piece discussed above. The change of the NH2 subdomain, as a result of inducer binding, results in a 3.5 angstrom displacement of the alpha carbon of residue 62: this amino acid is the last amino acid of the head-piece, connecting the hinge region to the core protein molecule. The alteration of this residue, results in disruption in the interaction of the hinge helices, a freeing of the HTH bound DNA, and a reduction in the affinity for the operator by the lac repressor molecule. This is similar to the allosteric changes in the oxy form of hemoglobin. Specifically, in the induced state, there are electrostatic alterations between chains in a dimer in the following residues: Lys84 and Glu100, Gln117 and Arg118, and His 74 and Asp 278 <>. The changes in binding between these residues is a result of a 3 angstrom change in the positioning of their alpha carbon. D. Role of The Tetramere If the only allosteric changes in the repressor molecule occure between chains in a dimer, the question arises as to why there is a need for the tetramere structure at all. A single repressor molecule binds to two operators that are between 93 and 401 bp apart. One suggested mechanism is that the repressor dimers bind to separate operator sequences, forcing the DNA to conform to a loop structure. In order for this kind of DNA interaction to occur, there must be a sufficient length between the operator sequences, in addition to certain properties of the DNA. The formation of this loop structure makes DNA polymerase binding even more unfavorable than if only one repressor dimer were bound to the DNA. It is aparent that this mechanism has evolved in cells in order to completely shut off lac operon transcription, when the inducer is bound. This is most likely a result of the extreme importance of metabolism regulation in cells. Another possible mechanism for bending the DNA is to place another DNA binding protein to the DNA loop region causing a sharper bend to the loop. Cap interacts with the operator sequence between the first and second operator in order to induce a 900 bend in the DNA over 30 bp. It has been shown that the CAP repressor combination binds DNA tighter than the repressor molecule alone. This works out well because the production of CAP is increased at times in the cell when energy needs to be conserved, and lactose cleavage needs to be slowed. Chime Index References Lewis, M., Chang G., et al., 1996. "Crystal structure of teh Lactose Operon Repressor and Its Complexes with DNA and Inducer." Science 271:1247-1254. Lamerichs, R.M.J.N., et al., 1989. "H NMR study of a Comples between the lac Repressor Headpiece and a 22 Base Pair Symmetric lac Operator." Biochemistry 28:2985-2991. Friedman, Alan M., et al., 1995. "Crystal Structure of lac Repressor Core Tetramere and Its Implications for DNA looping." Science 268:1721-1727. Slijper, M. et al., 1997. "Backbone and Side Chain Dynamics of lac Repressor Headpiece (1-56) and Its Complex with DNA." Biochemistry 36:249-254. Branden, Carl and John Tooze, Introduction to Protein Structure,Garland Publishing; New York, 1991. Alberts, Bruce et al., Molecular Biology of the Cell: third edition, Garland Publishing Inc., New York, 1994. Lewin, Benjamin, Genes VI, Oxford University press, New York, 1997. Internet Site--for loop figure- http://www.med.upenn.edu/biocbiop/..._lab/ people/kercher/research2.html
The DNA fragments bound to the tetramere, are separated by about 25 angstroms. In addition, there are no interaction between the two pieces of DNA.
The inducer molecule, in this case an IPTG molecule, is bound to the lac repressor at the two protein core sub domains <>. The galactoside, IPTG is used as the inducer instead of a lactose molecule, because lactose can be degraded in the cell, while IPTG can not. Due to the IPTG molecule being semi-symmetric, it can interact with the repressor in one of two ways. The repressor has the opportunity to make three or four bonds to the hydroxyl groups of the IPTG inducer molecule. Specifically, interactions through four hydrogen bonds: Asn246, with O2 of IPTG, Arg197 with O3 and O4, and Asp149 with O6. In addition, the Trp220 amino acid is in van der walls contact with an isopropyl group of IPTG <>. There is also a hydrophobic "sugar pocket" with in the core protein, involving the amino acids: Leu73, Ala75, Pro76, Ile79, Trp220, and Phe293 <>. There are a collection of altered lac repressor molecules that can not elicit a release of the DNA upon binding. These molecules are either defective in sugar binding or at transmitting the allosteric signals to the protein binding domain. These mutant molecules are said to display the Is phenotype. Some of the mutations of these Is phenotype molecules are scattered around the molecule, however most are concentrated in five regions: residues 70-80, 90-100, 190-200, 245-250, and 272-277 <>. It is important to realize where these crippling mutations lie because these sites are integral for either inducer binding or allosteric signal transduction. Most of these sites are near the sugar binding site, with the exception of residues 90-100. For this reason, it is hypothesized that these residues are responsible for allosteric signaling. They form the first Beta sheet of the NH2 terminal subdomain of the core domain, located at the dimer interface. C. Allosteric Signal Transduction There are two different structures of the lac repressor molecule corresponding to the induced and repressed states (bound to DNA). It is thought that this change is most likely propagated through the hinge piece discussed above. The change of the NH2 subdomain, as a result of inducer binding, results in a 3.5 angstrom displacement of the alpha carbon of residue 62: this amino acid is the last amino acid of the head-piece, connecting the hinge region to the core protein molecule. The alteration of this residue, results in disruption in the interaction of the hinge helices, a freeing of the HTH bound DNA, and a reduction in the affinity for the operator by the lac repressor molecule. This is similar to the allosteric changes in the oxy form of hemoglobin. Specifically, in the induced state, there are electrostatic alterations between chains in a dimer in the following residues: Lys84 and Glu100, Gln117 and Arg118, and His 74 and Asp 278 <>. The changes in binding between these residues is a result of a 3 angstrom change in the positioning of their alpha carbon. D. Role of The Tetramere If the only allosteric changes in the repressor molecule occure between chains in a dimer, the question arises as to why there is a need for the tetramere structure at all. A single repressor molecule binds to two operators that are between 93 and 401 bp apart. One suggested mechanism is that the repressor dimers bind to separate operator sequences, forcing the DNA to conform to a loop structure. In order for this kind of DNA interaction to occur, there must be a sufficient length between the operator sequences, in addition to certain properties of the DNA. The formation of this loop structure makes DNA polymerase binding even more unfavorable than if only one repressor dimer were bound to the DNA. It is aparent that this mechanism has evolved in cells in order to completely shut off lac operon transcription, when the inducer is bound. This is most likely a result of the extreme importance of metabolism regulation in cells. Another possible mechanism for bending the DNA is to place another DNA binding protein to the DNA loop region causing a sharper bend to the loop. Cap interacts with the operator sequence between the first and second operator in order to induce a 900 bend in the DNA over 30 bp. It has been shown that the CAP repressor combination binds DNA tighter than the repressor molecule alone. This works out well because the production of CAP is increased at times in the cell when energy needs to be conserved, and lactose cleavage needs to be slowed. Chime Index References Lewis, M., Chang G., et al., 1996. "Crystal structure of teh Lactose Operon Repressor and Its Complexes with DNA and Inducer." Science 271:1247-1254. Lamerichs, R.M.J.N., et al., 1989. "H NMR study of a Comples between the lac Repressor Headpiece and a 22 Base Pair Symmetric lac Operator." Biochemistry 28:2985-2991. Friedman, Alan M., et al., 1995. "Crystal Structure of lac Repressor Core Tetramere and Its Implications for DNA looping." Science 268:1721-1727. Slijper, M. et al., 1997. "Backbone and Side Chain Dynamics of lac Repressor Headpiece (1-56) and Its Complex with DNA." Biochemistry 36:249-254. Branden, Carl and John Tooze, Introduction to Protein Structure,Garland Publishing; New York, 1991. Alberts, Bruce et al., Molecular Biology of the Cell: third edition, Garland Publishing Inc., New York, 1994. Lewin, Benjamin, Genes VI, Oxford University press, New York, 1997. Internet Site--for loop figure- http://www.med.upenn.edu/biocbiop/..._lab/ people/kercher/research2.html
There are a collection of altered lac repressor molecules that can not elicit a release of the DNA upon binding. These molecules are either defective in sugar binding or at transmitting the allosteric signals to the protein binding domain. These mutant molecules are said to display the Is phenotype. Some of the mutations of these Is phenotype molecules are scattered around the molecule, however most are concentrated in five regions: residues 70-80, 90-100, 190-200, 245-250, and 272-277 <>. It is important to realize where these crippling mutations lie because these sites are integral for either inducer binding or allosteric signal transduction. Most of these sites are near the sugar binding site, with the exception of residues 90-100. For this reason, it is hypothesized that these residues are responsible for allosteric signaling. They form the first Beta sheet of the NH2 terminal subdomain of the core domain, located at the dimer interface. C. Allosteric Signal Transduction There are two different structures of the lac repressor molecule corresponding to the induced and repressed states (bound to DNA). It is thought that this change is most likely propagated through the hinge piece discussed above. The change of the NH2 subdomain, as a result of inducer binding, results in a 3.5 angstrom displacement of the alpha carbon of residue 62: this amino acid is the last amino acid of the head-piece, connecting the hinge region to the core protein molecule. The alteration of this residue, results in disruption in the interaction of the hinge helices, a freeing of the HTH bound DNA, and a reduction in the affinity for the operator by the lac repressor molecule. This is similar to the allosteric changes in the oxy form of hemoglobin. Specifically, in the induced state, there are electrostatic alterations between chains in a dimer in the following residues: Lys84 and Glu100, Gln117 and Arg118, and His 74 and Asp 278 <>. The changes in binding between these residues is a result of a 3 angstrom change in the positioning of their alpha carbon. D. Role of The Tetramere If the only allosteric changes in the repressor molecule occure between chains in a dimer, the question arises as to why there is a need for the tetramere structure at all. A single repressor molecule binds to two operators that are between 93 and 401 bp apart. One suggested mechanism is that the repressor dimers bind to separate operator sequences, forcing the DNA to conform to a loop structure. In order for this kind of DNA interaction to occur, there must be a sufficient length between the operator sequences, in addition to certain properties of the DNA. The formation of this loop structure makes DNA polymerase binding even more unfavorable than if only one repressor dimer were bound to the DNA. It is aparent that this mechanism has evolved in cells in order to completely shut off lac operon transcription, when the inducer is bound. This is most likely a result of the extreme importance of metabolism regulation in cells. Another possible mechanism for bending the DNA is to place another DNA binding protein to the DNA loop region causing a sharper bend to the loop. Cap interacts with the operator sequence between the first and second operator in order to induce a 900 bend in the DNA over 30 bp. It has been shown that the CAP repressor combination binds DNA tighter than the repressor molecule alone. This works out well because the production of CAP is increased at times in the cell when energy needs to be conserved, and lactose cleavage needs to be slowed. Chime Index References Lewis, M., Chang G., et al., 1996. "Crystal structure of teh Lactose Operon Repressor and Its Complexes with DNA and Inducer." Science 271:1247-1254. Lamerichs, R.M.J.N., et al., 1989. "H NMR study of a Comples between the lac Repressor Headpiece and a 22 Base Pair Symmetric lac Operator." Biochemistry 28:2985-2991. Friedman, Alan M., et al., 1995. "Crystal Structure of lac Repressor Core Tetramere and Its Implications for DNA looping." Science 268:1721-1727. Slijper, M. et al., 1997. "Backbone and Side Chain Dynamics of lac Repressor Headpiece (1-56) and Its Complex with DNA." Biochemistry 36:249-254. Branden, Carl and John Tooze, Introduction to Protein Structure,Garland Publishing; New York, 1991. Alberts, Bruce et al., Molecular Biology of the Cell: third edition, Garland Publishing Inc., New York, 1994. Lewin, Benjamin, Genes VI, Oxford University press, New York, 1997. Internet Site--for loop figure- http://www.med.upenn.edu/biocbiop/..._lab/ people/kercher/research2.html
There are two different structures of the lac repressor molecule corresponding to the induced and repressed states (bound to DNA). It is thought that this change is most likely propagated through the hinge piece discussed above. The change of the NH2 subdomain, as a result of inducer binding, results in a 3.5 angstrom displacement of the alpha carbon of residue 62: this amino acid is the last amino acid of the head-piece, connecting the hinge region to the core protein molecule. The alteration of this residue, results in disruption in the interaction of the hinge helices, a freeing of the HTH bound DNA, and a reduction in the affinity for the operator by the lac repressor molecule. This is similar to the allosteric changes in the oxy form of hemoglobin.
Specifically, in the induced state, there are electrostatic alterations between chains in a dimer in the following residues: Lys84 and Glu100, Gln117 and Arg118, and His 74 and Asp 278 <>. The changes in binding between these residues is a result of a 3 angstrom change in the positioning of their alpha carbon. D. Role of The Tetramere If the only allosteric changes in the repressor molecule occure between chains in a dimer, the question arises as to why there is a need for the tetramere structure at all. A single repressor molecule binds to two operators that are between 93 and 401 bp apart. One suggested mechanism is that the repressor dimers bind to separate operator sequences, forcing the DNA to conform to a loop structure. In order for this kind of DNA interaction to occur, there must be a sufficient length between the operator sequences, in addition to certain properties of the DNA. The formation of this loop structure makes DNA polymerase binding even more unfavorable than if only one repressor dimer were bound to the DNA. It is aparent that this mechanism has evolved in cells in order to completely shut off lac operon transcription, when the inducer is bound. This is most likely a result of the extreme importance of metabolism regulation in cells. Another possible mechanism for bending the DNA is to place another DNA binding protein to the DNA loop region causing a sharper bend to the loop. Cap interacts with the operator sequence between the first and second operator in order to induce a 900 bend in the DNA over 30 bp. It has been shown that the CAP repressor combination binds DNA tighter than the repressor molecule alone. This works out well because the production of CAP is increased at times in the cell when energy needs to be conserved, and lactose cleavage needs to be slowed. Chime Index References Lewis, M., Chang G., et al., 1996. "Crystal structure of teh Lactose Operon Repressor and Its Complexes with DNA and Inducer." Science 271:1247-1254. Lamerichs, R.M.J.N., et al., 1989. "H NMR study of a Comples between the lac Repressor Headpiece and a 22 Base Pair Symmetric lac Operator." Biochemistry 28:2985-2991. Friedman, Alan M., et al., 1995. "Crystal Structure of lac Repressor Core Tetramere and Its Implications for DNA looping." Science 268:1721-1727. Slijper, M. et al., 1997. "Backbone and Side Chain Dynamics of lac Repressor Headpiece (1-56) and Its Complex with DNA." Biochemistry 36:249-254. Branden, Carl and John Tooze, Introduction to Protein Structure,Garland Publishing; New York, 1991. Alberts, Bruce et al., Molecular Biology of the Cell: third edition, Garland Publishing Inc., New York, 1994. Lewin, Benjamin, Genes VI, Oxford University press, New York, 1997. Internet Site--for loop figure- http://www.med.upenn.edu/biocbiop/..._lab/ people/kercher/research2.html
D. Role of The Tetramere
If the only allosteric changes in the repressor molecule occure between chains in a dimer, the question arises as to why there is a need for the tetramere structure at all. A single repressor molecule binds to two operators that are between 93 and 401 bp apart. One suggested mechanism is that the repressor dimers bind to separate operator sequences, forcing the DNA to conform to a loop structure. In order for this kind of DNA interaction to occur, there must be a sufficient length between the operator sequences, in addition to certain properties of the DNA. The formation of this loop structure makes DNA polymerase binding even more unfavorable than if only one repressor dimer were bound to the DNA. It is aparent that this mechanism has evolved in cells in order to completely shut off lac operon transcription, when the inducer is bound. This is most likely a result of the extreme importance of metabolism regulation in cells.
Another possible mechanism for bending the DNA is to place another DNA binding protein to the DNA loop region causing a sharper bend to the loop. Cap interacts with the operator sequence between the first and second operator in order to induce a 900 bend in the DNA over 30 bp. It has been shown that the CAP repressor combination binds DNA tighter than the repressor molecule alone. This works out well because the production of CAP is increased at times in the cell when energy needs to be conserved, and lactose cleavage needs to be slowed.
Lewis, M., Chang G., et al., 1996. "Crystal structure of teh Lactose Operon Repressor and Its Complexes with DNA and Inducer." Science 271:1247-1254.
Lamerichs, R.M.J.N., et al., 1989. "H NMR study of a Comples between the lac Repressor Headpiece and a 22 Base Pair Symmetric lac Operator." Biochemistry 28:2985-2991.
Friedman, Alan M., et al., 1995. "Crystal Structure of lac Repressor Core Tetramere and Its Implications for DNA looping." Science 268:1721-1727.
Slijper, M. et al., 1997. "Backbone and Side Chain Dynamics of lac Repressor Headpiece (1-56) and Its Complex with DNA." Biochemistry 36:249-254.
Branden, Carl and John Tooze, Introduction to Protein Structure,Garland Publishing; New York, 1991.
Alberts, Bruce et al., Molecular Biology of the Cell: third edition, Garland Publishing Inc., New York, 1994.
Lewin, Benjamin, Genes VI, Oxford University press, New York, 1997.
Internet Site--for loop figure-
http://www.med.upenn.edu/biocbiop/..._lab/ people/kercher/research2.html