Mutations in the occQ Operator That Decrease OccR-induced DNA Bending Do Not Cause Constitutive Promoter Activity

Abstract

OccR is a LysR-type transcriptional regulator ofAgrobacterium tumefaciens that positively regulates the octopine catabolism operon of the Ti plasmid. Positive control of theocc genes occurs in response to octopine, a metabolite released from plant tumors. Octopine causes DNA-bound OccR to undergo a conformational change from an inactive to an active state; this change is marked by a decrease in footprint length from 55 to 45 nucleotides as well as a relaxation of a high angle DNA bend. In this study, we first used gel filtration chromatography to show that OccR is dimeric in solution, and we used gel shift assays to show that OccR is tetrameric when bound to DNA. We then created a series of site-directed mutations in the OccR-binding site. Some mutations were designed to lock OccR-DNA complexes into a conformation resembling the inactive conformation, whereas other mutations were designed to lock complexes into the active conformation. These mutations altered the conformation of OccR-DNA complexes and their responses to octopine in ways that we had predicted. As expected, operator mutations that locked complexes into a conformation having a long footprint and a high angle DNA bend blocked activation by octopine in vivo. Surprisingly, however, mutations that lock OccR into a short footprint and low angle DNA bend failed to cause the protein to function constitutively. Furthermore, some of the latter mutations interfered with activation by octopine. We conclude that locking OccR into a conformation having a short footprint is not sufficient to cause constitutive activation, and octopine must cause at least one additional conformational change in the protein. OccR is a LysR-type transcriptional regulator ofAgrobacterium tumefaciens that positively regulates the octopine catabolism operon of the Ti plasmid. Positive control of theocc genes occurs in response to octopine, a metabolite released from plant tumors. Octopine causes DNA-bound OccR to undergo a conformational change from an inactive to an active state; this change is marked by a decrease in footprint length from 55 to 45 nucleotides as well as a relaxation of a high angle DNA bend. In this study, we first used gel filtration chromatography to show that OccR is dimeric in solution, and we used gel shift assays to show that OccR is tetrameric when bound to DNA. We then created a series of site-directed mutations in the OccR-binding site. Some mutations were designed to lock OccR-DNA complexes into a conformation resembling the inactive conformation, whereas other mutations were designed to lock complexes into the active conformation. These mutations altered the conformation of OccR-DNA complexes and their responses to octopine in ways that we had predicted. As expected, operator mutations that locked complexes into a conformation having a long footprint and a high angle DNA bend blocked activation by octopine in vivo. Surprisingly, however, mutations that lock OccR into a short footprint and low angle DNA bend failed to cause the protein to function constitutively. Furthermore, some of the latter mutations interfered with activation by octopine. We conclude that locking OccR into a conformation having a short footprint is not sufficient to cause constitutive activation, and octopine must cause at least one additional conformational change in the protein. maltose-binding protein dithiothreitol nucleotide The Agrobacterium tumefaciens OccR protein is a transcriptional regulator that activates the occQ operon of the Ti plasmid in response to octopine, a plant tumor-released arginine derivative (1Habeeb L.F. Wang L. Winans S.C. Mol. Plant-Microbe Interact. 1991; 4: 279-385Crossref Scopus (50) Google Scholar). This operon encodes proteins required for the uptake and catabolism of octopine (2Dessaux Y. Petit A. Tempe J. Verma D.P.S. Molecular Signals in Plant-Microbe Communications. CRC Press, Inc., Boca Raton, FL1992: 109-136Google Scholar), which serves as a source of carbon, nitrogen, and energy for tumor-colonizing bacteria. The occoperon also encodes the TraR protein, which is a quorum-sensing transcriptional regulator of the Ti plasmid tra regulon (3Fuqua C. Winans S.C. Mol. Microbiol. 1996; 20: 1199-1210Crossref PubMed Scopus (73) Google Scholar). OccR has a molecular mass of 32.7 kDa and is a member of the LysR family of DNA-binding transcriptional regulators (4Henikoff S.G. Haughn J.M. Calvo J.M. Wallace J.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6602-6606Crossref PubMed Scopus (373) Google Scholar), the largest known family of DNA-binding regulatory proteins in Proteobacteria (5Perez-Rueda E. Collado-Vides J. Nucleic Acids Res. 2000; 28: 1838-1847Crossref PubMed Scopus (220) Google Scholar). Sequence comparisons among LysR proteins indicate that they share a highly conserved amino-terminal domain that contains a helix-turn-helix DNA-binding motif and a far less conserved carboxyl-terminal domain that generally binds low molecular weight inducing ligands. LysR proteins are often encoded by genes that are transcribed divergently from their target genes, and these proteins often activate a target operon and simultaneously repress their own expression by binding to a single intergenic region (6Schell M. Annu. Rev. Microbiol. 1993; 47: 597-626Crossref PubMed Scopus (863) Google Scholar).OccR binds specific DNA sequences that lie upstream of the promoter it activates, PoccQ. In the absence of octopine, OccR protects a region from −80 to −28 nucleotides upstream of its target promoter. Octopine has little effect on binding affinity and does not alter the oligomeric state of bound OccR. However, octopine shortens the DNase I footprint by one helical turn such that only the region from −80 to −38 is protected. Furthermore, apo-OccR incites a high angle DNA bend at this binding site, and this bend angle is relaxed by the addition of octopine (7Wang L. Helmann J.D. Winans S.C. Cell. 1992; 69: 659-667Abstract Full Text PDF PubMed Scopus (97) Google Scholar). Both OccR conformations repress the divergentoccR promoter, but activation of the occ operon occurs only when OccR is in the latter conformation.DNA bending has been suggested to be an important event in transcriptional activation for a number of LysR family proteins (8Hryniewicz M.M. Kredich N.M. J. Bacteriol. 1995; 177: 2343-2353Crossref PubMed Google Scholar, 9Toledano M.B. Kullik I. Trinh F. Baird P.T. Schneider T.D. Storz G. Cell. 1994; 78: 897-909Abstract Full Text PDF PubMed Scopus (318) Google Scholar, 10Parsek M.R. Kivisaar M. Chakrabarty A.M. Mol. Microbiol. 1995; 15: 819-828Crossref PubMed Scopus (42) Google Scholar). Several have been shown to incite bends in their target promoters. For example, CysB of Salmonella typhimuriuminduces a bend at two promoters, and these bends are partially relieved by the inducer, N-acetyl-l-serine (8Hryniewicz M.M. Kredich N.M. J. Bacteriol. 1995; 177: 2343-2353Crossref PubMed Google Scholar). Similarly, the OxyR protein of Escherichia coli causes a high angle bend at the oxyR-oxyS intergenic region that is relieved by reactive oxygen species such as hydrogen peroxide (9Toledano M.B. Kullik I. Trinh F. Baird P.T. Schneider T.D. Storz G. Cell. 1994; 78: 897-909Abstract Full Text PDF PubMed Scopus (318) Google Scholar). CatR of Pseudomonas putida also causes a high angle DNA bend at the catBC promoter, whereas the addition of the inducercis,cis-muconate relaxes this bend (10Parsek M.R. Kivisaar M. Chakrabarty A.M. Mol. Microbiol. 1995; 15: 819-828Crossref PubMed Scopus (42) Google Scholar). Although in the case of OccR and the above proteins, the inducing ligands cause a relaxation of the DNA bend, this is not a general rule, and DNA bending patterns are dependent on the specific regulator. For instance, apo-CatR causes little if any bend at promoter but a bend in the of the inducer, to the binding of additional CatR (10Parsek M.R. Kivisaar M. Chakrabarty A.M. Mol. Microbiol. 1995; 15: 819-828Crossref PubMed Scopus (42) Google LysR proteins long of DNA upstream of their target long binding that LysR proteins are and have been to be in solution, are their oligomeric state when DNA bound on of LysR proteins, to be by the carboxyl-terminal region of the protein A. M.M. J. Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. Full Text PDF PubMed Google Scholar, I. J. M.B. Storz G. J. Bacteriol. 1995; 177: PubMed Google Scholar). Although binding to DNA is of the of an inducer, inducing ligands often cause a conformational change in these For some proteins, such as CatR M.R. Chakrabarty A.M. J. Bacteriol. 1992; PubMed Google Scholar), J. J. 1991; PubMed Scopus Google Scholar), and M. J. Bacteriol. PubMed Google Scholar), the inducing to the number of bound protein whereas in other LysR proteins, such as OccR (7Wang L. Helmann J.D. Winans S.C. Cell. 1992; 69: 659-667Abstract Full Text PDF PubMed Scopus (97) Google and OxyR (9Toledano M.B. Kullik I. Trinh F. Baird P.T. Schneider T.D. Storz G. Cell. 1994; 78: 897-909Abstract Full Text PDF PubMed Scopus (318) Google Scholar), the does not alter the number of bound but the of DNA the of its operator at L. Winans S.C. J. Mol. 1995; PubMed Scopus Google and L. Winans S.C. J. Mol. 1995; PubMed Scopus Google Scholar). and are sufficient and for high affinity DNA as long as additional sequences are to the and are the affinity little if any to binding affinity but are required for DNA bending L. Winans S.C. J. Mol. 1995; PubMed Scopus Google Scholar). The high affinity binding contains a that the motif to which LysR family M. M. Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google Scholar). This in the of the region by OccR and is to the by the OxyR protein (9Toledano M.B. Kullik I. Trinh F. Baird P.T. Schneider T.D. Storz G. Cell. 1994; 78: 897-909Abstract Full Text PDF PubMed Scopus (318) Google and share some to the high affinity whereas is that apo-OccR with and complexes not and the We that complexes binding at the the The is locked OccR into a conformation with a low angle DNA bend, whereas mutations and the relaxation of the DNA bend in the of octopine L. Winans S.C. J. Mol. 1995; PubMed Scopus Google Scholar), the effect of octopine at only one the study, we used site-directed of the DNA-binding to the for transcriptional activation at the DNA were on and with the high affinity binding at and We the of sequences in the DNA-binding on DNA and promoter in A. the of the of these we the of DNA bending in transcriptional activation at the an to OccR binds its operator we first to the of these that OccR is dimeric in and tetrameric when DNA-bound is with the at the occQ operator and with that OccR binds DNA a of Several other LysR family proteins were shown to be in For example, of of E. CatR of and of putida have been to be in J. Mol. 1993; PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, M.R. Chakrabarty A.M. J. Bacteriol. 1994; PubMed Google Scholar), whereas OxyR of E. CysB of S. of Pseudomonas and of putida have been shown to be in S. J. Full Text PDF PubMed Google Scholar, I. J. M.B. Storz G. J. Bacteriol. 1995; 177: PubMed Google Scholar, Kredich N.M. J. Full Text PDF PubMed Google Scholar, M. J. Bacteriol. 1991; PubMed Google Scholar). In some gel filtration at high protein which the protein into a oligomeric state in the only protein has been when bound to DNA is which binds as a M.M. Kredich N.M. J. Bacteriol. 1994; PubMed Google Scholar). Several other LysR proteins have been to as on the of the of their DNase I (7Wang L. Helmann J.D. Winans S.C. Cell. 1992; 69: 659-667Abstract Full Text PDF PubMed Scopus (97) Google that and a high affinity OccR-binding site, for one OccR and that the sequences which this site, are for high affinity In this study, we the that sequences at and sequences are and the of OccR to alter its conformation in response to octopine. This in that complexes this required high of octopine to cause a conformational to to had but These the that the a in the affinity of OccR for its operator and that these sequences at and cause OccR to at these it binds the by and and to and This had effect on conformation in the absence of octopine. However, low of octopine were sufficient to a conformational and a change in bend angle in the absence of octopine and a shift with as little as octopine. to a in that this to in the absence of octopine, but a bend angle in the of of octopine. We conclude that the not be by but that when a high affinity is created at this it be by and were mutations that of of complexes to have a low angle DNA bend at octopine and to be little by octopine. This that is for the OccR conformation that causes a high angle DNA bend and a long of and a high angle DNA bend at octopine We this to that is not for the high angle bend and long footprint that is for the low angle DNA bend and short had that operator mutations that OccR to the conformation having a low angle bend in cause it to the occQ promoter vivo. However, this not the as such were for had to in vivo. This is shown with conformation in the low angle DNA bend, but in is dependent on the of octopine and is only to the the Octopine must cause a conformational change in OccR that be by this operator a but in to octopine and However, at octopine the promoter in The is that this a in to octopine, in with but failed to expression at high octopine of with that and are for in the of octopine. of these mutations cause OccR to the conformation having a low angle bend, we that these also cause OccR to to only low of octopine. is that these it is that sequences at be is that this OccR in a conformation that the active conformation but from it in some that is for The Agrobacterium tumefaciens OccR protein is a transcriptional regulator that activates the occQ operon of the Ti plasmid in response to octopine, a plant tumor-released arginine derivative (1Habeeb L.F. Wang L. Winans S.C. Mol. Plant-Microbe Interact. 1991; 4: 279-385Crossref Scopus (50) Google Scholar). This operon encodes proteins required for the uptake and catabolism of octopine (2Dessaux Y. Petit A. Tempe J. Verma D.P.S. Molecular Signals in Plant-Microbe Communications. CRC Press, Inc., Boca Raton, FL1992: 109-136Google Scholar), which serves as a source of carbon, nitrogen, and energy for tumor-colonizing bacteria. The occoperon also encodes the TraR protein, which is a quorum-sensing transcriptional regulator of the Ti plasmid tra regulon (3Fuqua C. Winans S.C. Mol. Microbiol. 1996; 20: 1199-1210Crossref PubMed Scopus (73) Google Scholar). OccR has a molecular mass of 32.7 kDa and is a member of the LysR family of DNA-binding transcriptional regulators (4Henikoff S.G. Haughn J.M. Calvo J.M. Wallace J.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6602-6606Crossref PubMed Scopus (373) Google Scholar), the largest known family of DNA-binding regulatory proteins in Proteobacteria (5Perez-Rueda E. Collado-Vides J. Nucleic Acids Res. 2000; 28: 1838-1847Crossref PubMed Scopus (220) Google Scholar). Sequence comparisons among LysR proteins indicate that they share a highly conserved amino-terminal domain that contains a helix-turn-helix DNA-binding motif and a far less conserved carboxyl-terminal domain that generally binds low molecular weight inducing ligands. LysR proteins are often encoded by genes that are transcribed divergently from their target genes, and these proteins often activate a target operon and simultaneously repress their own expression by binding to a single intergenic region (6Schell M. Annu. Rev. Microbiol. 1993; 47: 597-626Crossref PubMed Scopus (863) Google Scholar). OccR binds specific DNA sequences that lie upstream of the promoter it activates, PoccQ. In the absence of octopine, OccR protects a region from −80 to −28 nucleotides upstream of its target promoter. Octopine has little effect on binding affinity and does not alter the oligomeric state of bound OccR. However, octopine shortens the DNase I footprint by one helical turn such that only the region from −80 to −38 is protected. Furthermore, apo-OccR incites a high angle DNA bend at this binding site, and this bend angle is relaxed by the addition of octopine (7Wang L. Helmann J.D. Winans S.C. Cell. 1992; 69: 659-667Abstract Full Text PDF PubMed Scopus (97) Google Scholar). Both OccR conformations repress the divergentoccR promoter, but activation of the occ operon occurs only when OccR is in the latter conformation. DNA bending has been suggested to be an important event in transcriptional activation for a number of LysR family proteins (8Hryniewicz M.M. Kredich N.M. J. Bacteriol. 1995; 177: 2343-2353Crossref PubMed Google Scholar, 9Toledano M.B. Kullik I. Trinh F. Baird P.T. Schneider T.D. Storz G. Cell. 1994; 78: 897-909Abstract Full Text PDF PubMed Scopus (318) Google Scholar, 10Parsek M.R. Kivisaar M. Chakrabarty A.M. Mol. Microbiol. 1995; 15: 819-828Crossref PubMed Scopus (42) Google Scholar). Several have been shown to incite bends in their target promoters. For example, CysB of Salmonella typhimuriuminduces a bend at two promoters, and these bends are partially relieved by the inducer, N-acetyl-l-serine (8Hryniewicz M.M. Kredich N.M. J. Bacteriol. 1995; 177: 2343-2353Crossref PubMed Google Scholar). Similarly, the OxyR protein of Escherichia coli causes a high angle bend at the oxyR-oxyS intergenic region that is relieved by reactive oxygen species such as hydrogen peroxide (9Toledano M.B. Kullik I. Trinh F. Baird P.T. Schneider T.D. Storz G. Cell. 1994; 78: 897-909Abstract Full Text PDF PubMed Scopus (318) Google Scholar). CatR of Pseudomonas putida also causes a high angle DNA bend at the catBC promoter, whereas the addition of the inducercis,cis-muconate relaxes this bend (10Parsek M.R. Kivisaar M. Chakrabarty A.M. Mol. Microbiol. 1995; 15: 819-828Crossref PubMed Scopus (42) Google Scholar). Although in the case of OccR and the above proteins, the inducing ligands cause a relaxation of the DNA bend, this is not a general rule, and DNA bending patterns are dependent on the specific regulator. For instance, apo-CatR causes little if any bend at promoter but a bend in the of the inducer, to the binding of additional CatR (10Parsek M.R. Kivisaar M. Chakrabarty A.M. Mol. Microbiol. 1995; 15: 819-828Crossref PubMed Scopus (42) Google Scholar). LysR proteins long of DNA upstream of their target long binding that LysR proteins are and have been to be in solution, are their oligomeric state when DNA bound on of LysR proteins, to be by the carboxyl-terminal region of the protein A. M.M. J. Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. Full Text PDF PubMed Google Scholar, I. J. M.B. Storz G. J. Bacteriol. 1995; 177: PubMed Google Scholar). Although binding to DNA is of the of an inducer, inducing ligands often cause a conformational change in these For some proteins, such as CatR M.R. Chakrabarty A.M. J. Bacteriol. 1992; PubMed Google Scholar), J. J. 1991; PubMed Scopus Google Scholar), and M. J. Bacteriol. PubMed Google Scholar), the inducing to the number of bound protein whereas in other LysR proteins, such as OccR (7Wang L. Helmann J.D. Winans S.C. Cell. 1992; 69: 659-667Abstract Full Text PDF PubMed Scopus (97) Google and OxyR (9Toledano M.B. Kullik I. Trinh F. Baird P.T. Schneider T.D. Storz G. Cell. 1994; 78: 897-909Abstract Full Text PDF PubMed Scopus (318) Google Scholar), the does not alter the number of bound but the of DNA OccR the of its operator at L. Winans S.C. J. Mol. 1995; PubMed Scopus Google and L. Winans S.C. J. Mol. 1995; PubMed Scopus Google Scholar). and are sufficient and for high affinity DNA as long as additional sequences are to the and are the affinity little if any to binding affinity but are required for DNA bending L. Winans S.C. J. Mol. 1995; PubMed Scopus Google Scholar). The high affinity binding contains a that the motif to which LysR family M. M. Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google Scholar). This in the of the region by OccR and is to the by the OxyR protein (9Toledano M.B. Kullik I. Trinh F. Baird P.T. Schneider T.D. Storz G. Cell. 1994; 78: 897-909Abstract Full Text PDF PubMed Scopus (318) Google Scholar). and share some to the high affinity whereas is that apo-OccR with and complexes not and the We that complexes binding at the the The is locked OccR into a conformation with a low angle DNA bend, whereas mutations and the relaxation of the DNA bend in the of octopine L. Winans S.C. J. Mol. 1995; PubMed Scopus Google Scholar), the effect of octopine at only one In the study, we used site-directed of the DNA-binding to the for transcriptional activation at the DNA were on and with the high affinity binding at and We the of sequences in the DNA-binding on DNA and promoter in A. the of the of these we the of DNA bending in transcriptional activation at the an to OccR binds its operator we first to the of these that OccR is dimeric in and tetrameric when DNA-bound is with the at the occQ operator and with that OccR binds DNA a of Several other LysR family proteins were shown to be in For example, of of E. CatR of and of putida have been to be in J. Mol. 1993; PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, M.R. Chakrabarty A.M. J. Bacteriol. 1994; PubMed Google Scholar), whereas OxyR of E. CysB of S. of Pseudomonas and of putida have been shown to be in S. J. Full Text PDF PubMed Google Scholar, I. J. M.B. Storz G. J. Bacteriol. 1995; 177: PubMed Google Scholar, Kredich N.M. J. Full Text PDF PubMed Google Scholar, M. J. Bacteriol. 1991; PubMed Google Scholar). In some gel filtration at high protein which the protein into a oligomeric state in the only protein has been when bound to DNA is which binds as a M.M. Kredich N.M. J. Bacteriol. 1994; PubMed Google Scholar). Several other LysR proteins have been to as on the of the of their DNase I (7Wang L. Helmann J.D. Winans S.C. Cell. 1992; 69: 659-667Abstract Full Text PDF PubMed Scopus (97) Google that and a high affinity OccR-binding site, for one OccR and that the sequences which this site, are for high affinity In this study, we the that sequences at and sequences are and the of OccR to alter its conformation in response to octopine. This in that complexes this required high of octopine to cause a conformational to to had but These the that the a in the affinity of OccR for its operator and that these sequences at and cause OccR to at these it binds the by and and to and This had effect on conformation in the absence of octopine. However, low of octopine were sufficient to a conformational and a change in bend angle in the absence of octopine and a shift with as little as octopine. to a in that this to in the absence of octopine, but a bend angle in the of of octopine. We conclude that the not be by but that when a high affinity is created at this it be by and were mutations that of of complexes to have a low angle DNA bend at octopine and to be little by octopine. This that is for the OccR conformation that causes a high angle DNA bend and a long of and a high angle DNA bend at octopine We this to that is not for the high angle bend and long footprint that is for the low angle DNA bend and short had that operator mutations that OccR to the conformation having a low angle bend in cause it to the occQ promoter vivo. However, this not the as such were for had to in vivo. This is shown with conformation in the low angle DNA bend, but in is dependent on the of octopine and is only to the the Octopine must cause a conformational change in OccR that be by this operator a but in to octopine and However, at octopine the promoter in The is that this a in to octopine, in with but failed to expression at high octopine of with that and are for in the of octopine. of these mutations cause OccR to the conformation having a low angle bend, we that these also cause OccR to to only low of octopine. is that these it is that sequences at be is that this OccR in a conformation that the active conformation but from it in some that is for In an to OccR binds its operator we first to the of these that OccR is dimeric in and tetrameric when DNA-bound is with the at the occQ operator and with that OccR binds DNA a of Several other LysR family proteins were shown to be in For example, of of E. CatR of and of putida have been to be in J. Mol. 1993; PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, M.R. Chakrabarty A.M. J. Bacteriol. 1994; PubMed Google Scholar), whereas OxyR of E. CysB of S. of Pseudomonas and of putida have been shown to be in S. J. Full Text PDF PubMed Google Scholar, I. J. M.B. Storz G. J. Bacteriol. 1995; 177: PubMed Google Scholar, Kredich N.M. J. Full Text PDF PubMed Google Scholar, M. J. Bacteriol. 1991; PubMed Google Scholar). In some gel filtration at high protein which the protein into a oligomeric state in the only protein has been when bound to DNA is which binds as a M.M. Kredich N.M. J. Bacteriol. 1994; PubMed Google Scholar). Several other LysR proteins have been to as on the of the of their DNase I We (7Wang L. Helmann J.D. Winans S.C. Cell. 1992; 69: 659-667Abstract Full Text PDF PubMed Scopus (97) Google that and a high affinity OccR-binding site, for one OccR and that the sequences which this site, are for high affinity In this study, we the that sequences at and sequences are and the of OccR to alter its conformation in response to octopine. This in that complexes this required high of octopine to cause a conformational to to had but These the that the a in the affinity of OccR for its operator and that these sequences at and cause OccR to at these it binds the The by and and to and This had effect on conformation in the absence of octopine. However, low of octopine were sufficient to a conformational and a change in bend angle in the absence of octopine and a shift with as little as octopine. to a in that this to in the absence of octopine, but a bend angle in the of of octopine. We conclude that the not be by but that when a high affinity is created at this it be by OccR. and were mutations that of of complexes to have a low angle DNA bend at octopine and to be little by octopine. This that is for the OccR conformation that causes a high angle DNA bend and a long of and a high angle DNA bend at octopine We this to that is not for the high angle bend and long footprint that is for the low angle DNA bend and short We had that operator mutations that OccR to the conformation having a low angle bend in cause it to the occQ promoter vivo. However, this not the as such were for had to in vivo. This is shown with conformation in the low angle DNA bend, but in is dependent on the of octopine and is only to the the Octopine must cause a conformational change in OccR that be by this operator a but in to octopine and However, at octopine the promoter in The is that this a in to octopine, in with but failed to expression at high octopine of with that and are for in the of octopine. of these mutations cause OccR to the conformation having a low angle bend, we that these also cause OccR to to only low of octopine. is that these it is that sequences at be is that this OccR in a conformation that the active conformation but from it in some that is for We are to for with site-directed and to for TraR protein and DNA