Although quite common in the eukaryotic cell, bacterial proteins with an extensive coiled-coil domain are still relatively rare. One of the few thus far documented examples, TlpA from Salmonella typhimurium, is characterized by a remarkably long (250 amino acids) α-helical coiled-coil domain. Herein, we demonstrate that TlpA is a novel, sequence-specific DNA-binding protein. Several tlpA deletion mutants have been constructed, and their corresponding protein products were purified and tested for DNA binding. Two of the mutant proteins were shown to be deficient in DNA binding. Both mutants were analyzed by circular dichroism and electron microscopy, supporting the notion that mutant proteins were largely intact despite lacking the amino acid residues necessary for DNA binding. In vivo studies with transcriptional tlpA-lacZ fusions demonstrated that TlpA acts as a repressor. Using the repressor phenotype as a readout, the chain exchange previously described in vitro could also be confirmed in vivo. We believe the coiled-coil domain acts not only as a dimerization interface but could also serve a role as a flexible modulator of the protein-DNA interaction. Although quite common in the eukaryotic cell, bacterial proteins with an extensive coiled-coil domain are still relatively rare. One of the few thus far documented examples, TlpA from Salmonella typhimurium, is characterized by a remarkably long (250 amino acids) α-helical coiled-coil domain. Herein, we demonstrate that TlpA is a novel, sequence-specific DNA-binding protein. Several tlpA deletion mutants have been constructed, and their corresponding protein products were purified and tested for DNA binding. Two of the mutant proteins were shown to be deficient in DNA binding. Both mutants were analyzed by circular dichroism and electron microscopy, supporting the notion that mutant proteins were largely intact despite lacking the amino acid residues necessary for DNA binding. In vivo studies with transcriptional tlpA-lacZ fusions demonstrated that TlpA acts as a repressor. Using the repressor phenotype as a readout, the chain exchange previously described in vitro could also be confirmed in vivo. We believe the coiled-coil domain acts not only as a dimerization interface but could also serve a role as a flexible modulator of the protein-DNA interaction. INTRODUCTIONThe α-helical coiled-coil motif has been widely described(1.Cohen C. Parry D.A.D. Proteins. 1990; 7: 1-15Crossref PubMed Scopus (665) Google Scholar, 2.Cohen C. Parry D.A.D. Science. 1994; 263: 488-489Crossref PubMed Scopus (150) Google Scholar). Heptad amino acid repeats (a-b-c-d-e-f-g) are the hallmark of this structure which is driven by apolar residues buried in a interface formed by two (or more) α-helical chains in the coiled-coil structure(3.Crick F.H.C. Acta Crystallogr. 1953; 6: 689-697Crossref Google Scholar, 4.McLachlan A.D. Stewart M. J. Mol. Biol. 1975; 98: 293-304Crossref PubMed Scopus (567) Google Scholar). Positions a and d of the heptad form the characteristic 3-4 hydrophobic repeat, which has been identified in the primary sequence of more than 200 proteins (5.Lupas A. van Dyke M. Stock J. Science. 1991; 252: 1162-1164Crossref PubMed Scopus (3452) Google Scholar).Coiled-coils are also found as components of eukaryotic transcription factors(6.Baxevanis A.D. Vinson C.R. Curr. Opin. Gen. Dev. 1993; 3: 278-285Crossref PubMed Scopus (176) Google Scholar). In the eukaryotic bZip family of proteins, a coiled-coil motif of 3-4 heptads in length enables dimerization and positioning of the two polypeptide chains into a DNA binding unit(7.O'Shea E.K. Klemm J.D. Kim P.S. Alber T. Science. 1991; 254: 539-544Crossref PubMed Scopus (1273) Google Scholar, 8.O'Shea E.K. Rutkowski R. Kim P.S. Science. 1989; 243: 538-542Crossref PubMed Scopus (697) Google Scholar). The involvement of the leucine zipper coiled-coil is also a centerpiece of the basic region helix-loop-helix-zipper and the basic region helix-loop-helix structures(6.Baxevanis A.D. Vinson C.R. Curr. Opin. Gen. Dev. 1993; 3: 278-285Crossref PubMed Scopus (176) Google Scholar). It is now evident that the coiled-coil motif is not unique to the bZip proteins, but can also be found in transcription factors with homeodomain or zinc finger DNA-binding motifs(9.Sessa G. Morelli G. Ruberti I. EMBO J. 1993; 12: 3507-3517Crossref PubMed Scopus (215) Google Scholar, 10.Marmorstein R. Harrison S.C. Genes & Dev. 1994; 8: 2504-2512Crossref PubMed Scopus (149) Google Scholar).Gene regulators that utilize the coiled-coil motif appear to be less abundant in bacteria, and distinct families have yet to be recognized. To date, there are only a few documented examples of bacterial proteins per se, where the coiled-coil is a major structural feature (11.Lupas A. Muller S. Goldie K. Engel A.M. Engel A. Baumeister W. J. Mol. Biol. 1995; 248: 180-189Crossref PubMed Scopus (49) Google Scholar, 12.Fischetti V.A. Clin. Microbiol. Rev. 1989; 2: 285-290Crossref PubMed Scopus (628) Google Scholar, 13.Niki H. Imamura R. Kitaoka M. Yamanaka K. Ogura T. Hiraga S. EMBO J. 1992; 11: 5101-5109Crossref PubMed Scopus (162) Google Scholar, 14.Jagura-Burdzy G. Thomas C.T. J. Mol. Biol. 1992; 225: 651-660Crossref PubMed Scopus (38) Google Scholar, 15.Hurme R. Namork E. Nurmiaho-Lassila E.-L. Rhen M. J. Biol. Chem. 1994; 269: 10675-10682Abstract Full Text PDF PubMed Google Scholar). Recently, several bacterial DNA-binding proteins with the common helix-turn-helix element have been proposed to contain a leucine zipper-like dimerization motif(16.Maxon M.E. Wigboldus J. Brot N. Weissbach H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7076-7079Crossref PubMed Scopus (44) Google Scholar, 17.Noonan B. Trust T.J. Mol. Microbiol. 1995; 17: 379-386Crossref PubMed Scopus (16) Google Scholar, 18.Levchenko I. York D. Filutowicz M. Gene (Amst.). 1994; 145: 65-68Crossref PubMed Scopus (11) Google Scholar, 19.Sasse-Dwight S. Gralla J.D. Cell. 1990; 62: 945-954Abstract Full Text PDF PubMed Scopus (128) Google Scholar). Most of these bacterial examples however, lack biophysical evidence supporting the presence of a coiled-coil. Nevertheless, one cannot exclude the possibility that the leucine repeats, in these so-called zipper regions of the bacterial regulators, could mediate dimerization, if not by coiled-coil-like interaction, by way of another novel conformation. Indeed, the x-ray crystal structures of two other bacterial gene regulators, catabolite gene activator protein and the lac repressor, show that they contain short coiled-coil motifs enabling subunit interaction(20.Weber I.T. Steitz T.A. J. Mol. Biol. 1987; 198: 311-326Crossref PubMed Scopus (408) Google Scholar, 21.Friedman A.M. Fischmann T.O. Steitz T.A. Science. 1995; 268: 1721-1727Crossref PubMed Scopus (271) Google Scholar).The TlpA protein encoded by the Salmonella typhimurium virulence plasmid forms an elongated homodimer coiled-coil(15.Hurme R. Namork E. Nurmiaho-Lassila E.-L. Rhen M. J. Biol. Chem. 1994; 269: 10675-10682Abstract Full Text PDF PubMed Google Scholar, 22.Koski P. Saarilahti H. Sukupolvi S. Taira S. Riikonen P. Österlund K. Hurme R. Rhen M. J. Biol. Chem. 1992; 267: 12258-12265Abstract Full Text PDF PubMed Google Scholar). Here we show that TlpA has an ability to autoregulate its own gene by sequence-specific binding to its promoter DNA, an intriguing finding when one considers the sparse occurrence of extensive coiled-coils in bacterial proteins. As a first step toward dissecting the role of the coiled-coil domain in TlpA, we constructed a panel of mutant proteins lacking various portions of the reading frame. Purified mutant proteins were subjected to DNA binding and transcription assays. Based on these results we could localize the DNA-binding region, at the N terminus adjacent to the predicted coiled-coil. Evidence for in vivo chain exchange also points to TlpA's flexibility as a gene regulator.EXPERIMENTAL PROCEDURESPlasmid ConstructionMethods for DNA manipulation and transformation have been previously described(23.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). were as by the and of tlpA or were from R. Namork E. Nurmiaho-Lassila E.-L. Rhen M. J. Biol. Chem. 1994; 269: 10675-10682Abstract Full Text PDF PubMed Google Scholar, 22.Koski P. Saarilahti H. Sukupolvi S. Taira S. Riikonen P. Österlund K. Hurme R. Rhen M. J. Biol. Chem. 1992; 267: 12258-12265Abstract Full Text PDF PubMed Google Scholar). In the tlpA is the of the promoter of in the tlpA is in and from its and were by in tlpA the region of to with a chain residues The for were and by the in tlpA of and were by the and of tlpA in tlpA-lacZ transcription were on the a in the M. Riikonen P. Taira S. Mol. Microbiol. 1993; PubMed Scopus Google Scholar). To intact tlpA tlpA as a into and as a into to The tlpA region that in by S. typhimurium virulence plasmid DNA as a for and as a the into the the were as into the corresponding of were with gene into the in of the tlpA promoter in an to from the on a (5.Lupas A. van Dyke M. Stock J. Science. 1991; 252: 1162-1164Crossref PubMed Scopus (3452) Google Scholar, A. Muller S. Goldie K. Engel A.M. Engel A. Baumeister W. J. Mol. Biol. 1995; 248: 180-189Crossref PubMed Scopus (49) Google with a sequence and a of a and d with a of the proteins TlpA and were in were for the and as described P. Saarilahti H. Sukupolvi S. Taira S. Riikonen P. Österlund K. Hurme R. Rhen M. J. Biol. Chem. 1992; 267: 12258-12265Abstract Full Text PDF PubMed Google Scholar). mutant were purified to the previously P. Saarilahti H. Sukupolvi S. Taira S. Riikonen P. Österlund K. Hurme R. Rhen M. J. Biol. Chem. 1992; 267: 12258-12265Abstract Full Text PDF PubMed Google for TlpA, and which were purified on exchange by with more than As a for the other protein a with the and subjected in to P. Saarilahti H. Sukupolvi S. Taira S. Riikonen P. Österlund K. Hurme R. Rhen M. J. Biol. Chem. 1992; 267: 12258-12265Abstract Full Text PDF PubMed Google Scholar). The DNA binding when tested at in not and for or the were with J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). A the sequence with and A with and products were and with DNA were in and J. 1991; PubMed Scopus Google proteins in were with DNA in a binding in in a of to were at a of and DNA at DNA at at a were for at with a step of the of the which and were on a in were in for at the were and analyzed a the with the at per binding in binding at of The DNA for the by with one of the two the A and A for were and or analyzed by plasmid in transcription is a with a the gene from the S. typhimurium virulence M. Riikonen P. Taira S. Mol. Microbiol. 1993; PubMed Scopus Google Scholar). were in E. were from the and were on and with and as described in Molecular Cold Spring Harbor Laboratory, Cold Spring Harbor, were an of and mutant TlpA proteins were from to at in in these from to As an of we the of from the of acid with the A. and W. C. as the for were by amino acid R. H. S.C. PubMed Scopus Google of of protein and in were in to a protein of were to previously in The were on the for with for another and with for The were in a at with the of tlpA in tlpA to of P. Saarilahti H. Sukupolvi S. Taira S. Riikonen P. Österlund K. Hurme R. Rhen M. J. Biol. Chem. 1992; 267: 12258-12265Abstract Full Text PDF PubMed Google which an of to a role for TlpA to this protein have DNA-binding the DNA the tlpA gene by of two to two of two the or the Both were subjected to with purified protein. TlpA a of only the which the of the the tlpA gene and in the portions as for The that the other a that there is in the protein and also points to a binding with the tlpA gene from tlpA with or were and with TlpA or only the binding TlpA to a of the in protein the DNA by the finding that purified TlpA DNA we constructed and purified various TlpA mutant proteins for more of the interaction. proteins were that the residues of the protein sequence and the predicted coiled-coil sequence Using a (11.Lupas A. Muller S. Goldie K. Engel A.M. Engel A. Baumeister W. J. Mol. Biol. 1995; 248: 180-189Crossref PubMed Scopus (49) Google we can of coiled-coil which is shown to two of the protein the binding to the of the DNA we thus a this region for and in the mutant protein In a DNA the virulence also to sequence of the binding. The DNA only with the binding or TlpA binding The DNA the a distinct binding by TlpA as to a where protein binding also with more than a of the or DNA the to the binding. A mutant which amino in the a of DNA binding to to TlpA and the and of regions in TlpA amino acid deletion of TlpA are shown with the residues in protein. are in the proteins are in their but lacking the residues of the of coiled-coil is shown as a of amino acid in TlpA protein. and with the A. van Dyke M. Stock J. Science. 1991; 252: 1162-1164Crossref PubMed Scopus (3452) Google Scholar, A. Muller S. Goldie K. Engel A.M. Engel A. Baumeister W. J. Mol. Biol. 1995; 248: 180-189Crossref PubMed Scopus (49) Google assays. for TlpA binding. TlpA with the or DNA and subjected to a DNA were at more than to the binding. binding of TlpA mutant proteins to DNA with DNA DNA which the to the of with TlpA and The shown to with TlpA in the as a DNA also in the The mutant protein a from a only DNA, the that not with the acid and TlpA a region on and The on a promoter region, a element on the transcription previously P. Saarilahti H. Sukupolvi S. Taira S. Riikonen P. Österlund K. Hurme R. Rhen M. J. Biol. Chem. 1992; 267: 12258-12265Abstract Full Text PDF PubMed Google and a element where only residues are a is evident in the of the tlpA a DNA to and A to sequence and protein A sequence of the tlpA is shown where the region identified in is by a or the corresponding In the sequence the transcription is by a P. Saarilahti H. Sukupolvi S. Taira S. Riikonen P. Österlund K. Hurme R. Rhen M. J. Biol. Chem. 1992; 267: 12258-12265Abstract Full Text PDF PubMed Google and the the first in of with the that to with DNA in or we a to but with a region, constructed in to the residues for the protein-DNA more The mutant lacking amino evidence of binding to TlpA in and protein or or and the of in and protein as as the structural of the mutant proteins, as to TlpA, circular dichroism to of the as a for of The of and mutants are from the TlpA that their and their coiled-coil intact despite the dichroism of TlpA and the deletion and were from to at at in an of purified proteins these and in the binding on and to in the of the of mutant proteins were for their to form in electron TlpA as shown R. Namork E. Nurmiaho-Lassila E.-L. Rhen M. J. Biol. Chem. 1994; 269: 10675-10682Abstract Full Text PDF PubMed Google of where of not could be in the also a we could not of in the also to into of TlpA and the two mutants to a of and of TlpA to the of TlpA on its promoter in transcriptional fusions to the tlpA gene were constructed and shown that TlpA to promoter we this to have an on transcription as transcription of the fusions were in with an plasmid tlpA The only a of the tlpA reading that promoter could be from a of that the an of TlpA, a of as to that TlpA a repressor on the tlpA As a TlpA were in with a transcription with a plasmid TlpA from its own promoter or a promoter a when TlpA in of and at TlpA of the promoter of on the other not to supporting in vitro which a lack of region in that protein The of TlpA from a promoter or a promoter is by of E. TlpA from tlpA promoter cannot be in TlpA as by the of an TlpA in the R. Namork E. Nurmiaho-Lassila E.-L. Rhen M. J. Biol. Chem. 1994; 269: 10675-10682Abstract Full Text PDF PubMed Google Scholar). The for of TlpA to exclude the possibility of of of TlpA which previously has been shown to the with this R. Namork E. Nurmiaho-Lassila E.-L. Rhen M. J. Biol. Chem. 1994; 269: 10675-10682Abstract Full Text PDF PubMed Google in with an to TlpA chain exchange in vivo as has been demonstrated in R. Namork E. Nurmiaho-Lassila E.-L. Rhen M. J. Biol. Chem. 1994; 269: 10675-10682Abstract Full Text PDF PubMed Google Scholar). As an of we the transcription of TlpA in with We that if in a mutant TlpA a DNA-binding domain could the transcription by not of the tlpA Indeed, when in to the TlpA an of that from to protein from its promoter on a plasmid can also be in to the lack of the DNA-binding region and the to also that there is a of the protein for we have described that S. typhimurium virulence plasmid for a coiled-coil protein TlpA which is a as shown in in vitro and is also to form structures in R. Namork E. Nurmiaho-Lassila E.-L. Rhen M. J. Biol. Chem. 1994; 269: 10675-10682Abstract Full Text PDF PubMed Google Scholar, 22.Koski P. Saarilahti H. Sukupolvi S. Taira S. Riikonen P. Österlund K. Hurme R. Rhen M. J. Biol. Chem. 1992; 267: 12258-12265Abstract Full Text PDF PubMed Google Scholar). The of at yet finding that TlpA is a gene with the flexibility by the exchange sequence of binding by TlpA is demonstrated by and assays. to DNA, and could be only with a this sequence The to a in could that R. Namork E. Nurmiaho-Lassila E.-L. Rhen M. J. Biol. Chem. 1994; 269: 10675-10682Abstract Full Text PDF PubMed Google which the the The for this is that a DNA and with in the of TlpA be that the of be also a region of and identified the and in the more on the is to by TlpA or binding to several in the promoter panel we have demonstrated the of the DNA-binding region with to the coiled-coil domain and also to the role of the in the binding. The only residues deletion to DNA binding are that to the of TlpA, adjacent to the predicted coiled-coil as from lack of binding by and the coiled-coil adjacent to the DNA-binding region a in the as shown by and can be as a at the of or more as of binding more protein is per DNA, coiled-coil a role in positioning the binding an is by the that with a deletion in the coiled-coil but more to residues in a which in to that by the TlpA for can be as a element of for binding the DNA forms also a for Based on the phenotype of not we believe the binding structure to be a studies we can that to previously as the R. Namork E. Nurmiaho-Lassila E.-L. Rhen M. J. Biol. Chem. 1994; 269: 10675-10682Abstract Full Text PDF PubMed Google is to form with which has a deletion at the N a to TlpA and we believe also forms is into not the of the for of in electron It is yet that the to a in an of the with an but forms of In electron that the is and this not be in a polypeptide the in serve as a for of of the residues which are for DNA interaction. We not to the amino are of the residues to in the DNA-binding region and the DNA-binding residues form an domain or are an of the coiled-coil. studies are to the of the DNA-binding domain in TlpA, which could a novel of a DNA-binding structure to a coiled-coil. we are to to DNA-binding motifs in of tlpA that TlpA of a where the N terminus is for DNA binding and the adjacent long coiled-coil to the binding and for sequence have shown that the region by TlpA an promoter which can be by TlpA in but not by also the for for in vivo chain we shown that TlpA at are in exchange with the of this R. Namork E. Nurmiaho-Lassila E.-L. Rhen M. J. Biol. Chem. 1994; 269: 10675-10682Abstract Full Text PDF PubMed Google Scholar). The transcription which is by when in a which also the DNA-binding mutant is to by the tlpA can be if one considers that for TlpA from the there is to be a protein for or chain exchange be of one with an intact and one lacking this is to eukaryotic or proteins which have a and a DNA-binding and form with other and transcription factors from binding to their R. D. H. Cell. 1990; Full Text PDF PubMed Scopus Google Scholar, D. Genes & Dev. 1992; 6: PubMed Scopus Google Scholar). The in the of TlpA points also to the of the coiled-coil in the structure into a binding gene as TlpA have a role in the of as S. typhimurium, which is to its the and in the as from one to another its of in and Rev. Microbiol. 1995; PubMed Scopus Google Scholar). structures which are from studies to to in the A. P. J. Biol. Chem. 1991; Full Text PDF PubMed Google could be to in the or could the the coiled-coil domain and be by the can of TlpA there a in the cell, and by the the could be more toward the which could not DNA by studies are at the of and the for TlpA interaction. is to that a of gene regulators that the coiled-coil motif and for this protein family is the E. also characterized by an extensive coiled-coil domain and an ability to autoregulate G. Thomas C.T. J. Mol. Biol. 1992; 225: 651-660Crossref PubMed Scopus (38) Google Scholar). INTRODUCTIONThe α-helical coiled-coil motif has been widely described(1.Cohen C. Parry D.A.D. Proteins. 1990; 7: 1-15Crossref PubMed Scopus (665) Google Scholar, 2.Cohen C. Parry D.A.D. Science. 1994; 263: 488-489Crossref PubMed Scopus (150) Google Scholar). Heptad amino acid repeats (a-b-c-d-e-f-g) are the hallmark of this structure which is driven by apolar residues buried in a interface formed by two (or more) α-helical chains in the coiled-coil structure(3.Crick F.H.C. Acta Crystallogr. 1953; 6: 689-697Crossref Google Scholar, 4.McLachlan A.D. Stewart M. J. Mol. Biol. 1975; 98: 293-304Crossref PubMed Scopus (567) Google Scholar). Positions a and d of the heptad form the characteristic 3-4 hydrophobic repeat, which has been identified in the primary sequence of more than 200 proteins (5.Lupas A. van Dyke M. Stock J. Science. 1991; 252: 1162-1164Crossref PubMed Scopus (3452) Google Scholar).Coiled-coils are also found as components of eukaryotic transcription factors(6.Baxevanis A.D. Vinson C.R. Curr. Opin. Gen. Dev. 1993; 3: 278-285Crossref PubMed Scopus (176) Google Scholar). In the eukaryotic bZip family of proteins, a coiled-coil motif of 3-4 heptads in length enables dimerization and positioning of the two polypeptide chains into a DNA binding unit(7.O'Shea E.K. Klemm J.D. Kim P.S. Alber T. Science. 1991; 254: 539-544Crossref PubMed Scopus (1273) Google Scholar, 8.O'Shea E.K. Rutkowski R. Kim P.S. Science. 1989; 243: 538-542Crossref PubMed Scopus (697) Google Scholar). The involvement of the leucine zipper coiled-coil is also a centerpiece of the basic region helix-loop-helix-zipper and the basic region helix-loop-helix structures(6.Baxevanis A.D. Vinson C.R. Curr. Opin. Gen. Dev. 1993; 3: 278-285Crossref PubMed Scopus (176) Google Scholar). It is now evident that the coiled-coil motif is not unique to the bZip proteins, but can also be found in transcription factors with homeodomain or zinc finger DNA-binding motifs(9.Sessa G. Morelli G. Ruberti I. EMBO J. 1993; 12: 3507-3517Crossref PubMed Scopus (215) Google Scholar, 10.Marmorstein R. Harrison S.C. Genes & Dev. 1994; 8: 2504-2512Crossref PubMed Scopus (149) Google Scholar).Gene regulators that utilize the coiled-coil motif appear to be less abundant in bacteria, and distinct families have yet to be recognized. To date, there are only a few documented examples of bacterial proteins per se, where the coiled-coil is a major structural feature (11.Lupas A. Muller S. Goldie K. Engel A.M. Engel A. Baumeister W. J. Mol. Biol. 1995; 248: 180-189Crossref PubMed Scopus (49) Google Scholar, 12.Fischetti V.A. Clin. Microbiol. Rev. 1989; 2: 285-290Crossref PubMed Scopus (628) Google Scholar, 13.Niki H. Imamura R. Kitaoka M. Yamanaka K. Ogura T. Hiraga S. EMBO J. 1992; 11: 5101-5109Crossref PubMed Scopus (162) Google Scholar, 14.Jagura-Burdzy G. Thomas C.T. J. Mol. Biol. 1992; 225: 651-660Crossref PubMed Scopus (38) Google Scholar, 15.Hurme R. Namork E. Nurmiaho-Lassila E.-L. Rhen M. J. Biol. Chem. 1994; 269: 10675-10682Abstract Full Text PDF PubMed Google Scholar). Recently, several bacterial DNA-binding proteins with the common helix-turn-helix element have been proposed to contain a leucine zipper-like dimerization motif(16.Maxon M.E. Wigboldus J. Brot N. Weissbach H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7076-7079Crossref PubMed Scopus (44) Google Scholar, 17.Noonan B. Trust T.J. Mol. Microbiol. 1995; 17: 379-386Crossref PubMed Scopus (16) Google Scholar, 18.Levchenko I. York D. Filutowicz M. Gene (Amst.). 1994; 145: 65-68Crossref PubMed Scopus (11) Google Scholar, 19.Sasse-Dwight S. Gralla J.D. Cell. 1990; 62: 945-954Abstract Full Text PDF PubMed Scopus (128) Google Scholar). Most of these bacterial examples however, lack biophysical evidence supporting the presence of a coiled-coil. Nevertheless, one cannot exclude the possibility that the leucine repeats, in these so-called zipper regions of the bacterial regulators, could mediate dimerization, if not by coiled-coil-like interaction, by way of another novel conformation. Indeed, the x-ray crystal structures of two other bacterial gene regulators, catabolite gene activator protein and the lac repressor, show that they contain short coiled-coil motifs enabling subunit interaction(20.Weber I.T. Steitz T.A. J. Mol. Biol. 1987; 198: 311-326Crossref PubMed Scopus (408) Google Scholar, 21.Friedman A.M. Fischmann T.O. Steitz T.A. Science. 1995; 268: 1721-1727Crossref PubMed Scopus (271) Google Scholar).The TlpA protein encoded by the Salmonella typhimurium virulence plasmid forms an elongated homodimer coiled-coil(15.Hurme R. Namork E. Nurmiaho-Lassila E.-L. Rhen M. J. Biol. Chem. 1994; 269: 10675-10682Abstract Full Text PDF PubMed Google Scholar, 22.Koski P. Saarilahti H. Sukupolvi S. Taira S. Riikonen P. Österlund K. Hurme R. Rhen M. J. Biol. Chem. 1992; 267: 12258-12265Abstract Full Text PDF PubMed Google Scholar). Here we show that TlpA has an ability to autoregulate its own gene by sequence-specific binding to its promoter DNA, an intriguing finding when one considers the sparse occurrence of extensive coiled-coils in bacterial proteins. As a first step toward dissecting the role of the coiled-coil domain in TlpA, we constructed a panel of mutant proteins lacking various portions of the reading frame. Purified mutant proteins were subjected to DNA binding and transcription assays. Based on these results we could localize the DNA-binding region, at the N terminus adjacent to the predicted coiled-coil. Evidence for in vivo chain exchange also points to TlpA's flexibility as a gene
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Hurme et al. (1996) studied this question.