Identification and Characterization of the DNA-binding Domain of the Multifunctional PutA Flavoenzyme

The PutA flavoprotein from Escherichia coli is a transcriptional repressor and a bifunctional enzyme that regulates and catalyzes proline oxidation. PutA represses transcription of genes putA and putP by binding to the control DNA region of the put regulon. The objective of this study is to define and characterize the DNA binding domain of PutA. The DNA binding activity of PutA, a 1320 amino acid polypeptide, has been localized to N-terminal residues 1–261. After exploring a potential DNA-binding region and an N-terminal deletion mutant of PutA, residues 1–90 (PutA90) were determined to contain DNA binding activity and stabilize the dimeric structure of PutA. Cell-based transcriptional assays demonstrate that PutA90 functions as a transcriptional repressor in vivo. The dissociation constant of PutA90 with the put control DNA was estimated to be 110 nm, which is slightly higher than that of the PutA-DNA complex (Kd ∼ 45 nm). Primary and secondary structure analysis of PutA90 suggested the presence of a ribbon-helix-helix DNA binding motif in residues 1–47. To test this prediction, we purified and characterized PutA47. PutA47 is shown to purify as an apparent dimer, to exhibit in vivo transcriptional activity, and to bind specifically to the put control DNA. In gel-mobility shift assays, PutA47 was observed to bind cooperatively to the put control DNA with an overall dissociation constant of 15 nm for the PutA47-DNA complex. Thus, N-terminal residues 1–47 are critical for DNA-binding and the dimeric structure of PutA. These results are consistent with the ribbon-helix-helix family of transcription factors. The PutA flavoprotein from Escherichia coli is a transcriptional repressor and a bifunctional enzyme that regulates and catalyzes proline oxidation. PutA represses transcription of genes putA and putP by binding to the control DNA region of the put regulon. The objective of this study is to define and characterize the DNA binding domain of PutA. The DNA binding activity of PutA, a 1320 amino acid polypeptide, has been localized to N-terminal residues 1–261. After exploring a potential DNA-binding region and an N-terminal deletion mutant of PutA, residues 1–90 (PutA90) were determined to contain DNA binding activity and stabilize the dimeric structure of PutA. Cell-based transcriptional assays demonstrate that PutA90 functions as a transcriptional repressor in vivo. The dissociation constant of PutA90 with the put control DNA was estimated to be 110 nm, which is slightly higher than that of the PutA-DNA complex (Kd ∼ 45 nm). Primary and secondary structure analysis of PutA90 suggested the presence of a ribbon-helix-helix DNA binding motif in residues 1–47. To test this prediction, we purified and characterized PutA47. PutA47 is shown to purify as an apparent dimer, to exhibit in vivo transcriptional activity, and to bind specifically to the put control DNA. In gel-mobility shift assays, PutA47 was observed to bind cooperatively to the put control DNA with an overall dissociation constant of 15 nm for the PutA47-DNA complex. Thus, N-terminal residues 1–47 are critical for DNA-binding and the dimeric structure of PutA. These results are consistent with the ribbon-helix-helix family of transcription factors. Proline utilization A (PutA) from Escherichia coli is a multifunctional enzyme that catalyzes the flavin-dependent oxidation of proline to Δ1-pyrroline-5-carboxylate (P5C) 1The abbreviations used are: P5C, Δ1-pyrroline-5-carboxylate; PRODH, proline dehydrogenase; HTH, helix-turn-helix; RHH, ribbon-helix-helix; MES, 2-(N-morpholino)ethanesulfonic acid. and the NAD-dependent oxidation of P5C to glutamate (1Brown E. Wood J.M. J. Biol. Chem. 1992; 267: 13086-13092Google Scholar, 2Menzel R. Roth J. J. Biol. Chem. 1981; 256: 9762-9766Google Scholar). In the dehydrogenation of proline to P5C, two electrons are transferred from proline to a non-covalently bound FAD. Electrons from reduced FAD are then transferred to an acceptor in the electron transport chain to complete the catalytic cycle (3Abrahamson J.L.A. Baker L.G. Stephenson J.T. Wood J.M. Eur. J. Biochem. 1983; 134: 77-82Google Scholar). Next, P5C, the product of the first reaction, is hydrolyzed to glutamate-γ-semialdehyde followed by subsequent transfer of two electrons to an NAD+ cofactor by the P5C dehydrogenase domain to yield glutamate. In addition to enzymatic roles in proline utilization, PutA is responsible for the transcriptional regulation of the proline utilization genes putA and putP (1Brown E. Wood J.M. J. Biol. Chem. 1992; 267: 13086-13092Google Scholar, 4Menzel R. Roth J. J. Mol. Biol. 1981; 148: 21-44Google Scholar, 5Wood J.M. J. Bacteriol. 1981; 146: 895-901Google Scholar, 6Maloy S. Roth J.R. J. Bacteriol. 1983; 154: 561-568Google Scholar, 7Ostrovsky De Spicer P. O'Brian K. Maloy S. J. Bacteriol. 1991; 173: 211-219Google Scholar, 8Ostrovsky De Spicer P. Maloy S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4295-4298Google Scholar). PutP encodes a high affinity sodium-proline transporter (9Chen C.C. Tsuchiya T. Yamane Y. Wood J.M. Wilson T.H. J. Membr. Biol. 1985; 84: 157-164Google Scholar). PutA represses the expression of the put genes, which are transcribed in opposite directions, by binding to the put intergenic DNA region (1Brown E. Wood J.M. J. Biol. Chem. 1992; 267: 13086-13092Google Scholar, 7Ostrovsky De Spicer P. O'Brian K. Maloy S. J. Bacteriol. 1991; 173: 211-219Google Scholar, 8Ostrovsky De Spicer P. Maloy S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4295-4298Google Scholar). The transcriptional repression of the put genes is relieved in the presence of proline by the translocation of PutA to a peripheral position on the membrane, where proline is efficiently converted to glutamate. Reduction of the flavin causes a conformational change in PutA that is thought to enhance PutA membrane associations, thereby disrupting PutA-DNA binding (8Ostrovsky De Spicer P. Maloy S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4295-4298Google Scholar, 10Brown E.D. Wood J.M. J. Biol. Chem. 1993; 268: 8972-8979Google Scholar, 11Zhu W. Becker D.F. Biochemistry. 2003; 42: 5469-5477Google Scholar). Insights into the organization of the functional domains in PutA have been gained from molecular dissection and characterization of truncated PutA proteins. PutA is a polypeptide of 1320 amino acids and purifies predominately as a dimer, although minor amounts of monomer species are observed. The dimeric form of PutA has an estimated molecular mass of ∼293 kDa (1Brown E. Wood J.M. J. Biol. Chem. 1992; 267: 13086-13092Google Scholar). A truncated form of PutA containing residues 1–669 (PutA669) was shown to exhibit proline dehydrogenase (PRODH) and DNA binding activities but lack P5C dehydrogenase activity (12Vinod M.P. Bellur P. Becker D.F. Biochemistry. 2002; 41: 6525-6532Google Scholar). The x-ray crystal structure of PutA669 complexed to the competitive inhibitor l-lactate was solved to 2.0-Å resolution (13Lee Y.H. Nadaraia S. Gu D. Becker D.F. Tanner J.J. Nat. Struct. Biol. 2003; 10: 109-114Google Scholar). The crystal structure included residues 87–612 of PutA669 and revealed that the PRODH domain is a β8α8 barrel composed of residues 261–612, with the FAD bound at the C-terminal ends of the β-strands of the barrel. A putative helix-turn-helix (HTH) DNA-binding motif from residues 139–258 was also identified from the structure of the PutA669-lactate complex. The recognition helix (residues 230–241) of the HTH motif from the PutA669 structure contains basic residues that seem properly positioned for ionic and hydrogen bond interactions with DNA such as Arg-230, Arg-234, and Lys-238. PutA261, a truncated PutA protein containing residues 1–261, was shown to bind the put intergenic DNA region, demonstrating that DNA-binding activity can be separated from the PRODH domain and further implying that residues 139–258 were involved in DNA binding (13Lee Y.H. Nadaraia S. Gu D. Becker D.F. Tanner J.J. Nat. Struct. Biol. 2003; 10: 109-114Google Scholar). The goal of this study was to identify and characterize the DNA-binding domain of PutA. By engineering and examining several truncation and site-directed PutA mutants, we have identified the DNA-binding domain of PutA. In this report, we establish that N-terminal residues 1–47 comprise a domain that is responsible for the DNA binding activity of PutA. It is surprising that N-terminal residues 1–47 are also essential for dimerization of PutA, suggesting that this region is a key element for the versatility of PutA. The properties of the isolated DNA-binding domain (PutA47) are described and suggest that PutA is a member of the ribbon-helix-helix (RHH) family of DNA binding proteins. The implications of these findings are discussed in terms of the multifunctional properties of PutA. Materials—All chemicals and buffers were purchased from Fisher Scientific and Sigma-Aldrich, Inc. Restriction endonucleases and T4 DNA ligase were purchased from Fermentas and Promega, respectively. Molecular size standards used for calibrating size exclusion columns were purchased from Sigma. BCA reagents used for protein quantitation were obtained from Pierce. All experiments used Nano-pure water. E. coli strains XL-Blue and BL21 DE3 pLysS were purchased from Novagen. E. coli strain JT31 putA– lacZ– was a generous gift from J. Wood (University of Guelph, Guelph, ON, Canada). The vector pET-23b (Novagen) containing a hexahistidine-encoded sequence was used for expression of PutA669 R230/R234, PutAΔ85, and PutA261 as C-terminal hexahistidine-tagged proteins. The vector pET-14b was used for the expression of PutA139–258 as an N-terminal hexahistidine-tagged protein. The vector pET-3a (Novagen) was used for the expression of PutA90 and PutA47, which results in the addition of a GSGC amino acid sequence at the C-terminal end. Sequence-specific synthetic oligonucleotides were purchased from Integrated DNA The put intergenic DNA used for DNA binding assays was as described DNA from E. coli strain JT31 D.F. Biochemistry. Scholar). of PutA and truncation was by an at amino acid of the putA in pET-23b W. Y. P. Becker D.F. Biochem. 2002; Scholar). of the putA in PutA47 (residues and PutA90 (residues were by a at amino acid and of the putA in D.F. Biochemistry. Scholar). then the PutA amino acid in PutA47 and in The PutA139–258 (residues was by and at amino acid and of the putA in The containing residues 139–258 of PutA was into The of the and the was synthetic oligonucleotides and the site-directed A in PutA139–258 was from the of the The PutA669 mutant was in in pET-23b residues 1–669 of PutA with a C-terminal site-directed and oligonucleotides and for and (12Vinod M.P. Bellur P. Becker D.F. Biochemistry. 2002; 41: 6525-6532Google Scholar). acid of the PutA and PutA669 mutant A containing the control of the put region was as a of The obtained from a vector was positioned of the put control DNA region in vector containing the put intergenic DNA and to D.F. Biochemistry. Scholar). A of with and a that and the The was into and in a containing the control of the put control DNA to test in vivo transcriptional repressor activity of PutA, PutAΔ85, and PutA47 for were by the putA genes from into and Thus, of the PutA has a in the and were at the and of putA and the After with and the were into the putA genes of the acid of the the of putA and of PutA mutant PutAΔ85, PutA261, and PutA139–258 were in E. coli strain BL21 DE3 pLysS and purified a acid affinity (Novagen) as described for PutA669 and PutA261 (12Vinod M.P. Bellur P. Becker D.F. Biochemistry. 2002; 41: 6525-6532Google Scholar, Y.H. Nadaraia S. Gu D. Becker D.F. Tanner J.J. Nat. Struct. Biol. 2003; 10: 109-114Google Scholar). The were The purified were in containing at PutA90 and PutA47 were also in E. coli strain BL21 DE3 pLysS at as described but a (13Lee Y.H. Nadaraia S. Gu D. Becker D.F. Tanner J.J. Nat. Struct. Biol. 2003; 10: 109-114Google Scholar). PutA90 was purified by as described for PutA D.F. Biochemistry. Scholar). PutA90 was identified by analysis and gel-mobility shift PutA90 was then to a with and in the containing containing PutA90 were identified by analysis and into containing the for the PutA47 polypeptide is PutA47 was purified PutA47 was to a with MES, containing A from to in MES, and PutA47. containing PutA47 were identified by and gel-mobility shift PutA47 was then a with a molecular mass and purified further by a with PutA90 and PutA47 were in containing at The of the PutA were determined the BCA with as the and a at nm of for PutA669 mutant and and estimated at nm for PutA90 and PutA47 (12Vinod M.P. Bellur P. Becker D.F. Biochemistry. 2002; 41: 6525-6532Google Scholar, D.F. Biochemistry. Scholar). The for PutA90 and PutA47 at nm were determined by the of the in from to The of PutA90 and PutA47 was determined the of for PutA90 and PutA47. The at nm for PutA90 and PutA47 were estimated to be and respectively. PRODH activity was the at as described D.F. Biochemistry. Scholar). of PutA and PutA47 were from to nm in a an The was estimated from the at nm as described J. S. K. Biochemistry. Scholar). The molecular of PutAΔ85, and PutA47 were estimated by size exclusion was to a in containing and as described D.F. Biochemistry. Scholar). and PutA47 were to a in containing and the molecular mass standards dehydrogenase and DNA shift assays were used to test the binding of the PutA to the put control intergenic DNA as described D.F. Biochemistry. Scholar). The overall dissociation constant of PutA90 with the put intergenic DNA was estimated from binding The put intergenic DNA was at the with as described D.F. Biochemistry. Scholar). In a binding of a of PutA90 were with put intergenic DNA for 15 in The binding were then a and with of the binding After the were the on the from the was a The from binding containing DNA was used to for the of DNA to the at PutA90 were from experiments and by the of bound DNA PutA90 A dissociation constant was estimated by the to the where is the of DNA bound on the is the protein is the and is the of DNA In assays with PutA47, put intergenic DNA was used in the binding The synthetic with was used as of the in a to the put intergenic DNA. The put intergenic DNA was purified and by the acid at nm and the of the at nm an of to the of the PutA47 was with nm put intergenic DNA in a of in containing for DNA was to the binding to PutA47-DNA The PutA47-DNA were separated a at The were and the of DNA bound was determined a from binding experiments were and the coli strain JT31 putA– lacZ– containing and putA were at in with and to at nm ∼ were then and the protein from activity assays were in a of containing and The was determined by the in at of was by PRODH activity assays with the The results in are from activities from E. coli strain JT31 containing the and putA in a of DNA-binding to the N-terminal potential HTH motif was identified from the PutA669 x-ray crystal structure residues 139–258 (13Lee Y.H. Nadaraia S. Gu D. Becker D.F. Tanner J.J. Nat. Struct. Biol. 2003; 10: 109-114Google Scholar). A truncated PutA protein that contains the HTH motif was shown to bind specifically to the put control DNA To test the HTH motif was involved in DNA the HTH domain was and purified as to DNA binding activity in shift assays It was the lack of DNA binding was by by the of PutA139–258 to form a PutA139–258 purified as a polypeptide of molecular mass The molecular mass of PutA139–258 is that PutA139–258 is a To further the of the HTH domain in residues and were with in The of the two residues and apparent that and be involved in ionic interactions with the DNA and hydrogen bond interactions with (13Lee Y.H. Nadaraia S. Gu D. Becker D.F. Tanner J.J. Nat. Struct. Biol. 2003; 10: 109-114Google Scholar). of and Arg-234, the DNA binding activity of PutA669 PutA669 mutant has PRODH activity and an FAD with PutA669 of the Thus, from the results with PutA139–258 and PutA669 mutant that the putative HTH domain identified in the x-ray crystal structure of PutA669 was involved in DNA to the DNA-binding domain of PutA then on the N-terminal region of PutA. The of the N-terminal region in PutA-DNA binding was first by engineering the N-terminal deletion mutant PRODH activity of PutA and has two FAD at and nm in the with a of that is slightly higher than PutA D.F. Biochemistry. Scholar). contains of of polypeptide, to PutA of of by size exclusion a species with an estimated molecular mass of kDa is composed of amino acids the C-terminal and has a molecular mass of Thus, purifies as a monomer in to PutA, which purifies as a was also shown to lack DNA-binding was observed to bind the put control DNA at of to DNA the deletion of the N-terminal residues has on dimerization and DNA-binding activity of PutA. the DNA binding are purify as a The of to form a be the that DNA-binding the molecular dissection of PutA at amino acid a functional of PutA90 was then to test the of residues in DNA that PutA90 to the put control DNA demonstrating that the DNA binding activity is at the N-terminal region of PutA. PutA90 was estimated to have a molecular mass of PutA90 to a high molecular mass species the molecular mass of PutA90 is The estimated molecular mass of PutA90 contains residues involved in dimerization of PutA as observed with The overall dissociation constant of the complex was estimated by binding interactions apparent from the binding analysis of PutA90 to the put control DNA. A a and a of the binding was obtained by a A dissociation constant of nm was determined by the to the The for the complex is higher than the determined for the PutA-DNA complex (Kd ∼ 45 nm). The of PutA90 to bind put control DNA and transcription in vivo was assays in the E. coli strain JT31 putA– The was positioned of the put control DNA region in a vector truncated PutA were on a to test transcriptional repressor PutA reduced activity to to control that PutA protein. PutA669 and PutA90 expression with of activity to to In reduced activity to with control Thus, the assays demonstrate that PutA90 as a transcriptional repressor of the put control DNA in vivo. The repression of expression by PutA669 and PutA90 to PutA is by with the membrane, PutA with the DNA and the membrane (12Vinod M.P. Bellur P. Becker D.F. Biochemistry. 2002; 41: 6525-6532Google Scholar). of PutA the membrane of an structure of PutA90 was then for DNA binding A domain identified an domain of the family in PutA residues 1–47. the secondary structure of PutA47 characterized by a from residues and a domain from residues The of PutA47 with the sequence of the family in a sequence PutA47 and the is a transcriptional repressor of 45 amino acids P. A. A. R. A. J. Scholar). The DNA recognition element in the family is the which an in the complex to interactions P. A. A. R. A. J. Scholar, Scholar). The of the in the motif is to form key dimerization interactions P. A. A. R. A. J. Scholar). of the repressor of has shown that of residues in A and results in of the structure R. Biochemistry. Scholar, R. Struct. Biol. Scholar, R. Proc. Natl. Acad. Sci. U. S. A. Scholar). In PutA, and seem to be positioned as residues in a putative dimerization to the residues in PutA that are for the of the dimeric structure are in Thus, the motif contains involved in DNA-binding and dimerization that with that the DNA-binding and dimeric properties of PutA P. A. A. R. A. J. Scholar). of PutA with PutA47 is also shown in PutA is to have transcriptional repressor activity from and De Spicer P. O'Brian K. Maloy S. J. Bacteriol. 1991; 173: 211-219Google Scholar, Maloy S. J. Bacteriol. 1991; 173: Scholar, S. J. Scholar). In the such as and the regulation of the put has been on the in PutA to as a transcriptional repressor as The to identify an domain in PutA from such as and with these that PutA is involved in transcriptional regulation of the put genes S. Y. Scholar, P. J. Bacteriol. Scholar). Thus, structure PutA47 from E. coli be for PutA from is with DNA-binding PutA and the family is the lack of the helix A and helix A is in DNA-binding for a such as on the sequence than is to the helix A and in PutA. to be in PutA from A of has been on the of residues at the the of the A. D. Mol. 2003; Scholar). is to the of the PutA as a protein. PutA47 was purified to test the of the motif in PutA. of PutA and PutA47 are shown in the of at nm, the was estimated to be in PutA and in PutA47. The of in PutA47 of the which exhibit The molecular mass of PutA47 was estimated to be kDa by size exclusion the molecular mass of the PutA47 polypeptide is PutA47 purifies as a that PutA47 specifically to the put control DNA. A complex the of PutA47 in the DNA binding PutA47-DNA is also of dissociation of the PutA47-DNA complex of binding in the put control DNA. to PutA47 binding to the put control DNA A a and the binding were described a overall dissociation constant of 15 nm was estimated for the PutA47-DNA complex the PutA47 was also shown to as a transcriptional repressor in with activity to control Thus, characterization of PutA47 that residues 1–47 contain the DNA binding and dimerization domain of the PutA shift analysis of PutA47 DNA with of PutA47 nm to binding containing put control DNA and of DNA at The were separated a of of bound DNA PutA47 from the shift is the of the to the and to yield an overall dissociation constant of 15 and 15 nm but with and are shown for of the domain of the PutA flavoprotein is for multifunctional After of a putative DNA-binding domain in residues to the N-terminal region of PutA. The N-terminal deletion mutant was shown to lack DNA-binding activity and to purify as a suggesting the presence of a DNA-binding domain and a dimerization in the N-terminal region of PutA. characterization and domain analysis of PutA90 suggested that an DNA-binding domain was in residues 1–47. In a a dimerization is the β-strands form an that DNA. characterization of PutA47 that residues 1–47 contain the DNA binding domain of PutA and are involved in the dimeric structure of PutA. PutA47 specifically to the put control DNA region and as a transcriptional repressor in vivo. PutA47 also purifies as a and has consistent with the Thus, PutA is a the dimeric structure of PutA to on a dimerization to of the which are such as the repressor R. Biochemistry. Scholar). of the such as the dimeric and exhibit DNA binding on DNA P. A. A. R. A. J. Scholar, 1992; Scholar, Scholar). a also DNA cooperatively interactions A. R. Proc. Natl. Acad. Sci. U. S. A. Scholar). of the PutA47-DNA complex to consistent with properties of DNA-binding P. A. A. R. A. J. Scholar, A. R. Proc. Natl. Acad. Sci. U. S. A. Scholar). In with PutA, DNA binding has been although PutA binding in the put control DNA De Spicer P. O'Brian K. Maloy S. J. Bacteriol. 1991; 173: 211-219Google Scholar, M.P. Bellur P. Becker D.F. Biochemistry. 2002; 41: 6525-6532Google Scholar, D.F. Biochemistry. Scholar). analysis of PutA-DNA interactions be to the observed DNA binding of PutA47 is to the of PutA as a transcriptional repressor is to PutA47. of the PutA-DNA binding in the put control DNA region is in which of PutA-DNA with DNA have been observed to DNA In the x-ray crystal structure of the the DNA is bound by the P. A. A. R. A. J. Scholar). PutA binding has been shown to in the put control DNA De Spicer P. O'Brian K. Maloy S. J. Bacteriol. 1991; 173: 211-219Google Scholar). Thus, the DNA binding with PutA47 and the DNA observed with PutA are consistent with the DNA binding properties of the a of we PutA is a member of the family of transcriptional although of PutA as an member structure PutA the member of the family of with a of an motif on two catalytic of the family are from the repressor to the from E. with by C-terminal domains P. A. A. R. A. J. Scholar, P. R. Sci. Scholar). of C-terminal functional in protein in the binding of the in binding of the protein in the and binding by the repressor R. R. Nat. Struct. Biol. Scholar, Scholar, K. S. W. Biochem. J. 2002; Scholar, J. Biol. Chem. 1993; 268: Scholar, P. J. Biol. Chem. Scholar, E. Y. P. R. Nat. Struct. Biol. 2003; 10: Scholar). In a C-terminal domain is to an N-terminal E. Y. P. R. Nat. Struct. Biol. 2003; 10: Scholar). regulates in by binding to of the in the presence of to transcription of genes P. J. Biol. Chem. Scholar). In the of interactions are of the P. J. Biol. Chem. Scholar). of DNA-binding has been to conformational that are from the domain to the domain E. Y. P. R. Nat. Struct. Biol. 2003; 10: Scholar). PutA in which an domain is by a but in the of PutA, the regulation of DNA-binding FAD It that the motif is to regulation by The that the N-terminal region of PutA is responsible for DNA-binding activity and dimerization into the functional domain of PutA. The PRODH domain (residues has been by x-ray the of the P5C dehydrogenase domain (residues is on sequence analysis (13Lee Y.H. Nadaraia S. Gu D. Becker D.F. Tanner J.J. Nat. Struct. Biol. 2003; 10: 109-114Google Scholar, Wood J.M. J. Mol. Biol. Scholar). It is that of PutA by two polypeptide that at demonstrating an and the DNA-binding domain and the PRODH domain W. Becker D.F. Biochemistry. 2003; 42: 5469-5477Google Scholar). The of the region the DNA-binding and the PRODH domains is that conformational have been to residues in this region, specifically W. Becker D.F. Biochemistry. 2003; 42: 5469-5477Google Scholar). this region be involved in from the PRODH to the DNA-binding domain that PutA from a DNA-binding protein to a for PutA from a DNA-binding protein to a peripheral position on the membrane proline of FAD and a conformational change that the overall of PutA and (8Ostrovsky De Spicer P. Maloy S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4295-4298Google Scholar, 10Brown E.D. Wood J.M. J. Biol. Chem. 1993; 268: 8972-8979Google Scholar). Proline and FAD slightly the overall dissociation constant of the PutA-DNA complex D.F. Biochemistry. Scholar, Maloy S. Scholar). Maloy P. Maloy S. J. Bacteriol. have shown that in the presence of membrane proline PutA-DNA implying that the membrane has a critical on PutA-DNA the domain is on the domain of PutA to further the regulation of PutA (University of for with the binding

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