The Unstructured N-terminal Tail of ParG Modulates Assembly of a Quaternary Nucleoprotein Complex in Transcription Repression

Abstract

ParG is the prototype of a group of small (<10 kDa) proteins involved in accurate plasmid segregation. The protein is a dimeric DNA binding factor, which consists of symmetric paired C-terminal domains that interleave into a ribbon-helix-helix fold that is crucial for the interaction with DNA, and unstructured N-terminal domains of previously unknown function. Here the ParG protein is shown to be a transcriptional repressor of the parFG genes. The protein assembles on its operator site initially as a tetramer (dimer of dimers) and, at elevated protein concentrations, as a pair of tetramers. Progressive deletion of the mobile N-terminal tails concomitantly decreased transcriptional repression by ParG and perturbed the DNA binding kinetics of the protein. The flexible tails are not necessary for ParG dimerization but instead modulate the organization of a higher order nucleoprotein complex that is crucial for proper transcriptional repression. This is achieved by transient associations between the flexible and folded domains in complex with the target DNA. Numerous ParG homologs encoded by plasmids of Gram-negative bacteria similarly are predicted to possess N-terminal disordered tails, suggesting that this is a common feature of partition operon autoregulation. The results provide new insights into the role of natively unfolded domains in protein function, the molecular mechanisms of transcription regulation, and the control of plasmid segregation. ParG is the prototype of a group of small (<10 kDa) proteins involved in accurate plasmid segregation. The protein is a dimeric DNA binding factor, which consists of symmetric paired C-terminal domains that interleave into a ribbon-helix-helix fold that is crucial for the interaction with DNA, and unstructured N-terminal domains of previously unknown function. Here the ParG protein is shown to be a transcriptional repressor of the parFG genes. The protein assembles on its operator site initially as a tetramer (dimer of dimers) and, at elevated protein concentrations, as a pair of tetramers. Progressive deletion of the mobile N-terminal tails concomitantly decreased transcriptional repression by ParG and perturbed the DNA binding kinetics of the protein. The flexible tails are not necessary for ParG dimerization but instead modulate the organization of a higher order nucleoprotein complex that is crucial for proper transcriptional repression. This is achieved by transient associations between the flexible and folded domains in complex with the target DNA. Numerous ParG homologs encoded by plasmids of Gram-negative bacteria similarly are predicted to possess N-terminal disordered tails, suggesting that this is a common feature of partition operon autoregulation. The results provide new insights into the role of natively unfolded domains in protein function, the molecular mechanisms of transcription regulation, and the control of plasmid segregation. Plasmids are of inherent interest because of their contribution to bacterial genome plasticity, as model systems for investigating a variety of biological processes, and for their utility in gene cloning technology. The role of plasmids in the dissemination of antibiotic resistance and other properties is also particularly significant (1.Hayes F. Methods Mol. Biol. 2003; 235: 1-17PubMed Google Scholar). The events that contribute to stable plasmid inheritance can be unraveled by analyzing the molecular mechanisms that underlie plasmid segregation. The active segregation of low copy number plasmids typically requires two plasmid-encoded proteins, one of which is most commonly a member of the ParA superfamily of Walker-type ATPases. The second protein is a DNA binding factor that interacts directly with a cis-acting partition site (2.Surtees J.A. Funnell B.E. Curr. Top. Dev. Biol. 2003; 56: 145-180Crossref PubMed Scopus (45) Google Scholar). The assembly of the ParA- and DNA-binding proteins on the partition site engenders a nucleoprotein complex that is required for the directional movement of paired plasmids away from the cell median (3.Li Y. Dabrazhynetskaya A. Youngren B. Austin S. Mol. Microbiol. 2004; 53: 93-102Crossref PubMed Scopus (35) Google Scholar). Plasmid movement is most likely mediated by ATP-dependent polymerization of the ParA protein or by its depolymerization (4.Barillà D. Rosenberg M.F. Nobbmann U. Hayes F. EMBO J. 2005; 24: 1453-1464Crossref PubMed Scopus (118) Google Scholar). Unknown host factors are also probably required during segregation, for example as tethers for plasmid pairing at the mid-cell position. The partition locus of multidrug-resistance plasmid TP228 consists of the tandem parF and parG genes and additional essential sequences located upstream of these genes (5.Hayes F. Mol. Microbiol. 2000; 37: 528-541Crossref PubMed Scopus (75) Google Scholar). The ParF protein (22.0 kDa), a member of a distinctive subgroup of the ParA family, and ParG protein (8.6 kDa) assemble on the upstream region; ParG binds to this locus, and ParF is recruited through interactions with ParG, thereby forming a nucleoprotein complex whose precise architecture is likely to be crucial for correct partitioning (6.Barillà D. Hayes F. Mol. Microbiol. 2003; 49: 487-499Crossref PubMed Scopus (36) Google Scholar). ParG is dimeric (6.Barillà D. Hayes F. Mol. Microbiol. 2003; 49: 487-499Crossref PubMed Scopus (36) Google Scholar), consisting of a pair of unstructured N-terminal tails and intertwined, symmetric C-terminal domains that adopt a ribbon-helix-helix (RHH) 1The abbreviations used are: RHH, ribbon-helix-helix; CDO, catechol 2,3-dioxygenase; EMSA, electrophoretic mobility shift assay; IR, inverted repeat; HSQC, heteronuclear single quantum correlation. fold similar to that of the Arc/MetJ family of DNA binding transcriptional repressors (7.Golovanov A.P. Barillà D. Golovanova M. Hayes F. Lian L.Y. Mol. Microbiol. 2003; 50: 1141-1153Crossref PubMed Scopus (72) Google Scholar). The interaction of ParG with its binding sites in the region 5′ of parFG is presumed to occur via the double-stranded antiparallel β-structure of the RHH domain that is inserted into the DNA major groove in a manner analogous to Arc/MetJ-type transcriptional repressors (8.Somers W.S. Phillips S.E. Nature. 1992; 359: 387-393Crossref PubMed Scopus (284) Google Scholar, 9.Gomis-Ruth F.X. Sola M. Acebo P. Parraga A. Guasch A. Eritja R. Gonzalez A. Espinosa M. del Solar G. Coll M. EMBO J. 1998; 17: 7404-7415Crossref PubMed Scopus (135) Google Scholar, 10.Raumann B.E. Rould M.A. Pabo C.O. Sauer R.T. Nature. 1994; 367: 754-757Crossref PubMed Scopus (257) Google Scholar). Plasmid partition genes are organized in operons whose expression is precisely regulated. The control of partition gene expression is mediated, at least in part, by transcriptional autoregulation exerted by one or both of the partition proteins (11.Kusukawa N. Mori H. Kondo A. Hiraga S. Mol. Gen. Genet. 1987; 208: 365-372Crossref PubMed Scopus (38) Google Scholar, 12.Friedman S.A. Austin S.J. Plasmid. 1988; 19: 103-112Crossref PubMed Scopus (98) Google Scholar, 13.Hayes F. Radnedge L. Davis M.A. Austin S.J. Mol. Microbiol. 1994; 11: 249-260Crossref PubMed Scopus (55) Google Scholar, 14.Kwong S.M. Yeo C.C. Poh C.L. Mol. Microbiol. 2001; 40: 621-633Crossref PubMed Scopus (29) Google Scholar). The importance of this regulation is revealed by the perturbation that takes place in segregation when partition genes are overexpressed, especially when the partition site is coresident (11.Kusukawa N. Mori H. Kondo A. Hiraga S. Mol. Gen. Genet. 1987; 208: 365-372Crossref PubMed Scopus (38) Google Scholar, 13.Hayes F. Radnedge L. Davis M.A. Austin S.J. Mol. Microbiol. 1994; 11: 249-260Crossref PubMed Scopus (55) Google Scholar, 15.Funnell B.E. J. Bacteriol. 1988; 170: 954-960Crossref PubMed Google Scholar, 16.Davis M.A. Radnedge L. Martin K.A. Hayes F. Youngren B. Austin S.J. Mol. Microbiol. 1996; 21: 1029-1036Crossref PubMed Scopus (74) Google Scholar). Inappropriate production of partition proteins might result in their permanent occupation of the partition site, which could interfere with the migration of replication and transcription complexes (17.Youngren B. Austin S. Mol. Microbiol. 1997; 25: 1023-1030Crossref PubMed Scopus (31) Google Scholar, 18.Sawitzke J.A. Li Y. Sergueev K. Youngren B. Brendler T. Jones K. Austin S. J. Bacteriol. 2002; 184: 2447-2454Crossref PubMed Scopus (8) Google Scholar). The experiments presented here establish that the flexible N termini of dimeric ParG are essential for the organization of a stable quaternary protein complex on the parFG operator site and thus play a crucial regulatory role. This is achieved by transient associations between the flexible and folded domains in complex with the target DNA. Strains and Media—Escherichia coli at in with when with used for used for for protein and and S. A. J. Bacteriol. PubMed Google for Plasmids and parG gene previously by and in for ParG and in and for (6.Barillà D. Hayes F. Mol. Microbiol. 2003; 49: 487-499Crossref PubMed Scopus (36) Google Scholar). in which and from the of parG by and also in and proteins as for ParG (6.Barillà D. Hayes F. Mol. Microbiol. 2003; 49: 487-499Crossref PubMed Scopus (36) Google Scholar). DNA the parFG region and the of the parF and This in the site of plasmid T. 1997; PubMed Scopus Google to a transcriptional to the gene and of the and of the domain of to which transcriptional of the operon (6.Barillà D. Hayes F. Mol. Microbiol. 2003; 49: 487-499Crossref PubMed Scopus (36) Google Scholar). This results in on with at or when this operon is not G. J. A. D. U. S. A. 1998; PubMed Scopus Google Scholar). of ParG by the as previously B. Hayes F. 1997; 25: PubMed Scopus Google Scholar, F. B. Microbiol. 2000; PubMed Scopus Google Scholar, F. Genet. 2003; 37: PubMed Scopus Google with the of the of the on protein dimerization as and protein to the (6.Barillà D. Hayes F. Mol. Microbiol. 2003; 49: 487-499Crossref PubMed Scopus (36) Google Scholar). in a cell in at in at by and by to 1992; PubMed Scopus Google 2000; PubMed Scopus Google and to P. J. 2000; PubMed Scopus Google Scholar). coli with or or with and parG or in and at for additional and in with the cell by and protein the protein by the for at with at least in from the is the of that at J. PubMed Scopus Google Scholar). DNA a to the parFG operator a of this in which one of the and a one at for in binding with of ParG on in and DNA by and the used a The of the the and with The used in to the with the to of results between with to but with to one cell as a in at a of the of and and complexes and accurate protein in from to and with of the of The of the of the of the molecular and molecular of complexes from the K. 21: PubMed Scopus Google Scholar). in the region in a a of at ParG and in and at for the The of the complex by the of at of the DNA in and in complex with on a with a of ParG used and with protein at K. to the of at shift used proteins in and to protein and to the of protein A.P. S.A. Lian L.Y. J. 2004; PubMed Scopus Google Scholar). domain by F. S. G. J. A. J. PubMed Scopus Google Scholar), and and with J. 1994; PubMed Scopus Google Scholar). shift in and from deletion or DNA binding as shown in and are and from the of to of with in of in protein to of DNA that the of dimeric protein in one binding site to be on ParG the N-terminal can be in a on of in coli (6.Barillà D. Hayes F. Mol. Microbiol. 2003; 49: 487-499Crossref PubMed Scopus (36) Google Scholar, G. J. A. D. U. S. A. 1998; PubMed Scopus Google Scholar). of ParG with at by B. Hayes F. 1997; 25: PubMed Scopus Google Scholar, F. B. Microbiol. 2000; PubMed Scopus Google Scholar, F. Genet. 2003; 37: PubMed Scopus Google Scholar). of in the ParG N-terminal the with ParG in in the folded domain with ParG The the and of the ParG flexible but of the N-terminal involved and and on the of that in the of the N-terminal suggesting that this domain is not a (7.Golovanov A.P. Barillà D. Golovanova M. Hayes F. Lian L.Y. Mol. Microbiol. 2003; 50: 1141-1153Crossref PubMed Scopus (72) Google Scholar). The deletion both with ParG and in the as as ParG dimeric (6.Barillà D. Hayes F. Mol. Microbiol. 2003; 49: 487-499Crossref PubMed Scopus (36) Google Scholar). and similar a of revealed the of a single dimeric for of the proteins similar to results from of ParG (6.Barillà D. Hayes F. Mol. Microbiol. 2003; 49: 487-499Crossref PubMed Scopus (36) Google Scholar). the and that the N-terminal mobile of ParG role in ParG the of the parFG and with the parFG upstream of the parF is necessary for plasmid partitioning (5.Hayes F. Mol. Microbiol. 2000; 37: 528-541Crossref PubMed Scopus (75) Google and is by ParG (6.Barillà D. Hayes F. Mol. Microbiol. 2003; 49: 487-499Crossref PubMed Scopus (36) Google Scholar). The region upstream of the parF inverted that consists of by a the parFG to the of the The and for this to the for of with of the The the of the This organization that the is operator site in regulation of parFG the role of the upstream region in parFG a transcriptional by cloning a the IR, and of parF in a in plasmid This catechol with the low of the plasmid that the parFG is located in the region upstream of the parF The ParG protein from a expression and its in to a transcriptional repressor of the parFG of the from in the of to in the of ParG the role of the in this the of the to a similar to that in the in production from a the similar to that from the that is by the ParG expression from this results that ParG is a transcriptional repressor of parFG expression and that the is operator site required for this repression. The interaction between ParG and the region by with a the ParG two complexes and with this that into the at protein ParG not a of the located on a or on a on both of the not a in which the of a single complex with a migration similar to complex ParG not with of the in the by the of ParG to this ParG to the might be additional ParG on the The of ParG to by a of the K.A. PubMed Scopus Google for complexes K. 21: PubMed Scopus Google Scholar). and that the mobile of ParG its D. and this for as the of ParG in but of the of protein and complexes and between and or in The of the the of the The the molecular and the of the nucleoprotein complexes from the This molecular of and for complexes and are in with the for complexes by and two DNA and for and DNA The binding also by of at by the of ParG with with the DNA. the of that as a of the The that the a at a of ParG a complex similar to the previously complex The N-terminal for and the of ParG with and for repression of the to the unstructured transcriptional repression by ParG of the by a repression similar to that for ParG repression of the parFG for and which repression of and the unstructured of ParG a crucial role in transcriptional repression of the parFG and of the are by repression. The of the N-terminal in the interaction of ParG with the operator by and of binding to that similar to for complex most at low protein concentrations, with the of complex at higher and a of the into complex at the protein and interaction of with a with a migration from complexes or suggesting that the of with the particularly perturbed The of by experiments with with which the protein to a from the position. and both with with similar as ParG, complex in ParG, of the deletion proteins with experiments to the a a and with in in and of DNA as a in the at for and to for ParG and the N-terminal deletion to at a of and a similar at protein not the interaction with this target between the the with as to whose of ParG, and and from of which presented a particularly in the when the The in that ParG two complexes with that ParG and the N-terminal deletion a complex model in operator site of the by a for this complex from of binding for the of ParG and the N-terminal deletion to presented in the are in both and as to the by protein binding to This that kinetics the binding of ParG and its N-terminal deletion to the and in to the of binding to in binding to in the This might be because of between the and C-terminal of role of the N-terminal domain of ParG in complex with its operator site by the and C-terminal domains of ParG shift for ParG and its and of revealed that and in the of from as as the deletion site, suggesting that region not with the folded domain The by the which not the N the deletion site but also a significant number of J. 1994; PubMed Scopus Google Scholar, Solar G. 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Microbiol. 2003; 50: 1141-1153Crossref PubMed Scopus (72) Google Scholar). the N-terminal domain in the and complexes with DNA, significant in of from the flexible N of ParG, and not the and of of the N with are with and most the and of shift can be of complex the complex is stable the complex the protein. and the in between and from and not in the of the but from and with probably because of a in from and the C-terminal in the that deletion of the N-terminal domain not complex the of the complex used in which to the nucleoprotein complex and DNA and its on the in (7.Golovanov A.P. Barillà D. Golovanova M. Hayes F. Lian L.Y. Mol. Microbiol. 2003; 50: 1141-1153Crossref PubMed Scopus (72) Google Scholar). of ParG on DNA (7.Golovanov A.P. Barillà D. Golovanova M. Hayes F. Lian L.Y. Mol. Microbiol. 2003; 50: 1141-1153Crossref PubMed Scopus (72) Google Scholar). of to the the as from the of in from and the of for DNA to not from the folded domain of ParG and suggesting that to DNA and that ParG of the DNA from the of from the N-terminal domain that their to binding with to their shift in the the from and in the the that the N-terminal domain in DNA binding in a manner with the folded the N-terminal domain interaction not of the DNA the interactions are transient and with of DNA that are to the at the of the nucleoprotein ParG from by probably because this is a complex between ParG and DNA. the N-terminal of ParG to The RHH fold a of proteins that transcriptional repression by of a double-stranded antiparallel β-structure into the DNA major groove of their operator sites Solar G. F.X. Coll M. Espinosa M. J. Bacteriol. 2002; 184: PubMed Scopus Google Scholar). to their RHH C-terminal the and proteins mobile N-terminal tails that DNA and when the repressors to their target sites (8.Somers W.S. Phillips S.E. Nature. 1992; 359: 387-393Crossref PubMed Scopus (284) Google Scholar). tails provide additional binding in the of and Sauer R.T. U. S. A. PubMed Scopus Google Scholar). the flexible C-terminal region of the RHH protein is for binding with a M. K. S. J. 2002; PubMed Scopus (35) Google Scholar), and the disordered C-terminal of is required for binding W.S. Phillips Nature. PubMed Scopus Google Scholar). The RHH domain of the protein is by folded C-terminal that and binding Y. Sauer R.T. C.L. Biol. 2003; PubMed Scopus Google Scholar). major RHH proteins in their or C-terminal which are crucial for their biological function. RHH proteins, the of these to be The the that the RHH protein ParG is a transcriptional repressor of parFG expression and the operator The flexible N of ParG this repressor with thereby the of the protein. The disordered tails of ParG are not required for dimerization as deletion of the tails the interaction in or in unstructured proteins and protein domains are to a of the P. 2002; PubMed Scopus Google Scholar). The DNA binding and of unstructured proteins or protein for a variety of systems Curr. Biol. 2002; PubMed Scopus Google Scholar). of the and proteins in is mediated by the disordered C-terminal of of which to a that The complex a higher of and for its target operator site the proteins C.L. 1994; PubMed Scopus Google Scholar, T. PubMed Scopus Google Scholar). the binding in a disordered but a DNA This is by DNA that is crucial for the of other regulatory factors Li K. R. Nature. PubMed Scopus Google Scholar, Li J. 2004; PubMed Scopus Google Scholar). these and other proteins in which mobile domains DNA the N-terminal domain of ParG significant when on DNA. The in of from this domain between and that the interactions between the and DNA are number of in the are in these with a significant forming with the on the is a in the of the N-terminal domain with and the shift of in the N-terminal domain in the complex are in the complex with DNA, that the flexible N of ParG particularly to DNA binding to its operator site might in the C-terminal region of ParG that are to the mobile N-terminal This with other RHH proteins in which flexible that the when to the target away from the protein to on the DNA (8.Somers W.S. Phillips S.E. Nature. 1992; 359: 387-393Crossref PubMed Scopus (284) Google Scholar, 10.Raumann B.E. Rould M.A. Pabo C.O. Sauer R.T. Nature. 1994; 367: 754-757Crossref PubMed Scopus (257) Google Scholar, K. Phillips 1994; PubMed Scopus Google Scholar). of the mobile of ParG of most in in which of the through the and into the of the folded domain The in in the that fold into antiparallel that is predicted to be involved in DNA This is most likely for the properties by as the kinetics of binding to the operator site, of the interaction with the and the in transcriptional repression exerted by this protein. region of the N-terminal of ParG a to the that interacts with the DNA of the folded domain to two additional antiparallel ParG the of the folded domain of ParG is predicted to in the major groove of DNA, is to that the transient could the DNA-binding site in other the region of the flexible domain might play role. this the most between the folded and N-terminal domains of ParG and the DNA provide to the with the of the transient from the folded the for interaction with the binding the deletion of in and the of the DNA could the of this protein. The C-terminal folded domain of is and its transcriptional repression and DNA binding be by perturbation of the interaction between the unfolded and folded in to transient interactions with the DNA-binding domain via the disordered N-terminal tails of ParG might interactions the nucleoprotein thereby directly to the of a stable nucleoprotein complex at the operator is that the flexible N-terminal domain one or complex regulatory in the of ParG interactions with its target ParG two complexes with its target site that into a single at nucleoprotein complexes are with and on DNA for the and ParG not with operator but the of complex with a to which a is that one of the two this complex is probably on the to with the complex Sauer R.T. PubMed Scopus (55) Google Scholar). This the that ParG binds its sites as a of the binding of ParG to the operator site is a tetramer (dimer of dimers) a pair of in the operator that with of other RHH proteins in complex with DNA (8.Somers W.S. Phillips S.E. Nature. 1992; 359: 387-393Crossref PubMed Scopus (284) Google Scholar, 9.Gomis-Ruth F.X. Sola M. Acebo P. Parraga A. Guasch A. Eritja R. Gonzalez A. Espinosa M. del Solar G. Coll M. EMBO J. 1998; 17: 7404-7415Crossref PubMed Scopus (135) Google Scholar, 10.Raumann B.E. Rould M.A. Pabo C.O. Sauer R.T. Nature. 1994; 367: 754-757Crossref PubMed Scopus (257) Google Scholar). the complex in which a second tetramer is The deletion of or N-terminal not this of interactions in deletion of the N in of a complex with revealed that the binding properties of from of ParG of the complex interactions between ParG and additional factors as the binding sites are or and occur in the target site or protein to be in at the kinetics of ParG interactions with this revealed that the target site when to D. and F. Numerous plasmids ParG most of which are predicted to possess N-terminal mobile of (5.Hayes F. Mol. Microbiol. 2000; 37: 528-541Crossref PubMed Scopus (75) Google Scholar, A.P. Barillà D. Golovanova M. Hayes F. Lian L.Y. Mol. Microbiol. 2003; 50: 1141-1153Crossref PubMed Scopus (72) Google Scholar, Barillà D. Hayes F. J. Bacteriol. 2005; PubMed Scopus Google Scholar). the sequences of the are is likely that these domains the role in transcriptional repression as the N-terminal of transcriptional by the of a repressor protein a flexible is likely to be a common in partition operon autoregulation by in to its interaction with the IR, ParG binds to additional sequences that the cis-acting partition site upstream of D. and F. sequences of that are to the but are (6.Barillà D. Hayes F. Mol. Microbiol. 2003; 49: 487-499Crossref PubMed Scopus (36) Google Scholar). be to the role of the mobile N-terminal of ParG in the organization of the nucleoprotein complex at this and for to the for in for plasmid for to the and for on the