Guoling Tian, Ph.D.

Disease-associated mutations in TUBA1A result in a spectrum of defects in the tubulin folding and heterodimer assembly pathway
Tian, Guoling; Jaglin, Xavier H; Keays, David A; Francis, Fiona; Chelly, Jamel; Cowan, Nicholas J
2010 Sep 15;19(18):3599-3613, Human molecular genetics
Malformations of cortical development are characteristic of a plethora of diseases that includes polymicrogyria, periventricular and subcortical heterotopia and lissencephaly. Mutations in TUBA1A and TUBB2B, each a member of the multigene families that encode alpha- and beta-tubulins, have recently been implicated in these diseases. Here we examine the defects that result from nine disease-causing mutations (I188L, I238V, P263T, L286F, V303G, L397P, R402C, 402H, S419L) in TUBA1A. We show that the expression of all the mutant proteins in vitro results in the generation of tubulin heterodimers in varying yield and that these can co-polymerize with microtubules in vitro. We identify several kinds of defects that result from these mutations. Among these are various defects in the chaperone-dependent pathway leading to de novo tubulin heterodimer formation. These include a defective interaction with the chaperone prefoldin, a reduced efficiency in the generation of productive folding intermediates as a result of inefficient interaction with the cytosolic chaperonin, CCT, and, in several cases, a failure to stably interact with TBCB, one of five tubulin-specific chaperones that act downstream of CCT in the tubulin heterodimer assembly pathway. Other defects include structural instability in vitro, diminished stability in vivo, a compromised ability to co-assemble with microtubules in vivo and a suppression of microtubule growth rate in the neurites (but not the soma) of cultured neurons. Our data are consistent with the notion that some mutations in TUBA1A result in tubulin deficit, whereas others reflect compromised interactions with one or more MAPs that are essential to proper neuronal migration
— id: 112037, year: 2010, vol: 19, page: 3599, stat: Journal Article,

Effect of TBCD and its regulatory interactor Arl2 on tubulin and microtubule integrity
Tian, Guoling; Thomas, Simi; Cowan, Nicholas J
2010 Nov;67(11):706-714, Cytoskeleton (Hoboken)
Assembly of the alpha/beta tubulin heterodimer requires the participation of a series of chaperone proteins (TBCA-E) that function downstream of the cytosolic chaperonin (CCT) as a heterodimer assembly machine. TBCD and TBCE are also capable of acting in a reverse reaction in which they disrupt native heterodimers. Homologs of TBCA-E exist in all eukaryotes, and the amino acid sequences of alpha- and beta-tubulin isotypes are rigidly conserved among vertebrates. However, the efficiency with which TBCD effects tubulin disruption in vivo depends on its origin: bovine (but not human) TBCD efficiently destroys tubulin and microtubules upon overexpression in cultured cells. Here we show that recombinant bovine TBCD is produced in HeLa cells as a stoichiometric cocomplex with beta-tubulin, consistent with its behavior in vitro and in vivo. In contrast, expression of human TBCD using the same host/vector system results in the generation of TBCD that is not complexed with beta-tubulin. We show that recombinant human TBCD functions indistinguishably from its nonrecombinant bovine counterpart in in vitro CCT-driven folding reactions, in tubulin disruption reactions, and in tubulin GTPase activating protein assays in which TBCD and TBCC stimulate GTP hydrolysis by beta-tubulin at a heterodimer concentration far below that required for polymerization into microtubules. We conclude that bovine and human TBCD have functionally identical roles in de novo tubulin heterodimer assembly, and show that the inability of human TBCD to disrupt microtubule integrity upon overexpression in vivo can be overcome by siRNA-mediated suppression of expression of the TBCD regulator Arl2 (ADP ribosylation factor-like protein). (c) 2010 Wiley-Liss, Inc
— id: 113945, year: 2010, vol: 67, page: 706, stat: Journal Article,

Mutations in the beta-tubulin gene TUBB2B result in asymmetrical polymicrogyria
Jaglin, Xavier Hubert; Poirier, Karine; Saillour, Yoann; Buhler, Emmanuelle; Tian, Guoling; Bahi-Buisson, Nadia; Fallet-Bianco, Catherine; Phan-Dinh-Tuy, Francoise; Kong, Xiang Peng; Bomont, Pascale; Castelnau-Ptakhine, Laetitia; Odent, Sylvie; Loget, Philippe; Kossorotoff, Manoelle; Snoeck, Irina; Plessis, Ghislaine; Parent, Philippe; Beldjord, Cherif; Cardoso, Carlos; Represa, Alfonso; Flint, Jonathan; Keays, David Anthony; Cowan, Nicholas Justin; Chelly, Jamel
2009 Jun;41(6):746-752, Nature genetics
Polymicrogyria is a relatively common but poorly understood defect of cortical development characterized by numerous small gyri and a thick disorganized cortical plate lacking normal lamination. Here we report de novo mutations in a beta-tubulin gene, TUBB2B, in four individuals and a 27-gestational-week fetus with bilateral asymmetrical polymicrogyria. Neuropathological examination of the fetus revealed an absence of cortical lamination associated with the presence of ectopic neuronal cells in the white matter and in the leptomeningeal spaces due to breaches in the pial basement membrane. In utero RNAi-based inactivation demonstrates that TUBB2B is required for neuronal migration. We also show that two disease-associated mutations lead to impaired formation of tubulin heterodimers. These observations, together with previous data, show that disruption of microtubule-based processes underlies a large spectrum of neuronal migration disorders that includes not only lissencephaly and pachygyria, but also polymicrogyria malformations
— id: 135247, year: 2009, vol: 41, page: 746, stat: Journal Article,

A Pachygyria-causing {alpha}-Tubulin Mutation Results in Inefficient Cycling with CCT and a Deficient Interaction with TBCB
Tian, Guoling; Kong, Xiang-Peng; Jaglin, Xavier H; Chelly, Jamel; Keays, David; Cowan, Nicholas J
2008 Mar;19(3):1152-1161, Molecular biology of the cell
The agyria (lissencephaly)/pachygyria phenotypes are catastrophic developmental diseases characterized by abnormal folds on the surface of the brain and disorganized cortical layering. In addition to mutations in at least four genes-LIS1, DCX, ARX and RELN-mutations in a human alpha-tubulin gene, TUBA1A, have recently been identified that cause these diseases. Here, we show that one such mutation, R264C, leads to a diminished capacity of de novo tubulin heterodimer formation. We identify the mechanisms that contribute to this defect. First, there is a reduced efficiency whereby quasinative alpha-tubulin folding intermediates are generated via ATP-dependent interaction with the cytosolic chaperonin CCT. Second, there is a failure of CCT-generated folding intermediates to stably interact with TBCB, one of the five tubulin chaperones (TBCA-E) that participate in the pathway leading to the de novo assembly of the tubulin heterodimer. We describe the behavior of the R264C mutation in terms of its effect on the structural integrity of alpha-tubulin and its interaction with TBCB. In spite of its compromised folding efficiency, R264C molecules that do productively assemble into heterodimers are capable of copolymerizing into dynamic microtubules in vivo. The diminished production of TUBA1A tubulin in R264C individuals is consistent with haploinsufficiency as a cause of the disease phenotype
— id: 78375, year: 2008, vol: 19, page: 1152, stat: Journal Article,

Mutations in alpha-tubulin cause abnormal neuronal migration in mice and lissencephaly in humans
Keays, David A; Tian, Guoling; Poirier, Karine; Huang, Guo-Jen; Siebold, Christian; Cleak, James; Oliver, Peter L; Fray, Martin; Harvey, Robert J; Molnar, Zoltan; Pinon, Maria C; Dear, Neil; Valdar, William; Brown, Steve D M; Davies, Kay E; Rawlins, J Nicholas P; Cowan, Nicholas J; Nolan, Patrick; Chelly, Jamel; Flint, Jonathan
2007 Jan 12;128(1):45-57, Cell
The development of the mammalian brain is dependent on extensive neuronal migration. Mutations in mice and humans that affect neuronal migration result in abnormal lamination of brain structures with associated behavioral deficits. Here, we report the identification of a hyperactive N-ethyl-N-nitrosourea (ENU)-induced mouse mutant with abnormalities in the laminar architecture of the hippocampus and cortex, accompanied by impaired neuronal migration. We show that the causative mutation lies in the guanosine triphosphate (GTP) binding pocket of alpha-1 tubulin (Tuba1) and affects tubulin heterodimer formation. Phenotypic similarity with existing mouse models of lissencephaly led us to screen a cohort of patients with developmental brain anomalies. We identified two patients with de novo mutations in TUBA3, the human homolog of Tuba1. This study demonstrates the utility of ENU mutagenesis in the mouse as a means to discover the basis of human neurodevelopmental disorders
— id: 78376, year: 2007, vol: 128, page: 45, stat: Journal Article,

Cryptic out-of-frame translational initiation of TBCE rescues tubulin formation in compound heterozygous HRD
Tian, Guoling; Huang, Melissa C; Parvari, Ruti; Diaz, George A; Cowan, Nicholas J
2006 Sep 5;103(36):13491-13496, Proceedings of the National Academy of Sciences of the United States of America
Microtubules are indispensable dynamic structures that contribute to many essential biological functions. Assembly of the native alpha/beta tubulin heterodimer, the subunit that polymerizes to form microtubules, requires the participation of several molecular chaperones, namely prefoldin, the cytosolic chaperonin CCT, and a series of five tubulin-specific chaperones termed cofactors A-E (TBCA-E). Among these, TBCC, TBCD, and TBCE are essential in higher eukaryotes; they function together as a multimolecular machine that assembles quasinative CCT-generated alpha- and beta-tubulin polypeptides into new heterodimers. Deletion and truncation mutations in the gene encoding TBCE have been shown to cause the rare autosomal recessive syndrome known as HRD, a devastating disorder characterized by congenital hypoparathyroidism, mental retardation, facial dysmorphism, and extreme growth failure. Here we identify cryptic translational initiation at each of three out-of-frame AUG codons upstream of the genetic lesion as a unique mechanism that rescues a mutant HRD allele by producing a functional TBCE protein. Our data explain how afflicted individuals, who would otherwise lack the capacity to make functional TBCE, can survive and point to a limiting capacity to fold tubulin heterodimers de novo as a contributing factor to disease pathogenesis
— id: 67543, year: 2006, vol: 103, page: 13491, stat: Journal Article,

Mutations affecting beta-tubulin folding and degradation
Wang, Yaqing; Tian, Guoling; Cowan, Nicholas J; Cabral, Fernando
2006 May 12;281(19):13628-13635, Journal of biological chemistry
Revertants of a colcemid-resistant Chinese hamster ovary cell line with an altered (D45Y) beta-tubulin have allowed the identification of four cis-acting mutations (L187R, Y398C, a 12-amino acid in-frame deletion, and a C-terminal truncation) that act by destabilizing the mutant tubulin and preventing it from incorporating into microtubules. These unstable beta-tubulins fail to form heterodimers and are predominantly found in association with the chaperonin CCT, suggesting that they cannot undergo productive folding. In agreement with these in vivo observations, we show that the defective beta-tubulins do not stably interact with cofactors involved in the tubulin folding pathway and, hence, fail to exchange with beta-tubulin in purified alphabeta heterodimers. Treatment of cells with MG132 causes an accumulation of the aberrant tubulins, indicating that improperly folded beta-tubulin is degraded by the proteasome. Rapid degradation of the mutant tubulin does not elicit compensatory changes in wild-type tubulin synthesis or assembly. Instead, loss of beta-tubulin from the mutant allele causes a 30-40% decrease in cellular tubulin content with no obvious effect on cell growth or survival
— id: 67544, year: 2006, vol: 281, page: 13628, stat: Journal Article,

Identification of a novel tubulin-destabilizing protein related to the chaperone cofactor E
Bartolini, Francesca; Tian, Guoling; Piehl, Michelle; Cassimeris, Lynne; Lewis, Sally A; Cowan, Nicholas J
2005 Mar 15;118(Pt 6):1197-1207, Journal of cell science
Factors that regulate the microtubule cytoskeleton are critical in determining cell behavior. Here we describe the function of a novel protein that we term E-like based on its sequence similarity to the tubulin-specific chaperone cofactor E. We find that upon overexpression, E-like depolymerizes microtubules by committing tubulin to proteosomal degradation. Our data suggest that this function is direct and is based on the ability of E-like to disrupt the tubulin heterodimer in vitro. Suppression of E-like expression results in an increase in the number of stable microtubules and a tight clustering of endocellular membranes around the microtubule-organizing center, while the properties of dynamic microtubules are unaffected. These observations define E-like as a novel regulator of tubulin stability, and provide a link between tubulin turnover and vesicle transport
— id: 56011, year: 2005, vol: 118, page: 1197, stat: Journal Article,

Tubulin folding cofactors as GTPase-activating proteins. GTP hydrolysis and the assembly of the alpha/beta-tubulin heterodimer
Tian G; Bhamidipati A; Cowan NJ; Lewis SA
1999 Aug 20;274(34):24054-24058, Journal of biological chemistry
In vivo, many proteins must interact with molecular chaperones to attain their native conformation. In the case of tubulin, newly synthesized alpha- and beta-subunits are partially folded by cytosolic chaperonin, a double-toroidal ATPase with homologs in all kingdoms of life and in most cellular compartments. alpha- and beta-tubulin folding intermediates are then brought together by tubulin-specific chaperone proteins (named cofactors A-E) in a cofactor-containing supercomplex with GTPase activity. Here we show that tubulin subunit exchange can only occur by passage through this supercomplex, thus defining it as a dimer-making machine. We also show that hydrolysis of GTP by beta-tubulin in the supercomplex acts as a switch for the release of native tubulin heterodimer. In this folding reaction and in the related reaction of tubulin-folding cofactors with native tubulin, the cofactors behave as GTPase-activating proteins, stimulating the GTP-binding protein beta-tubulin to hydrolyze its GTP
— id: 6176, year: 1999, vol: 274, page: 24054, stat: Journal Article,

The alpha- and beta-tubulin folding pathways
Lewis, S A; Tian, G; Cowan, N J
1997 Dec;7(12):479-484, Trends in cell biology
The alpha-beta tubulin heterodimer is the subunit from which microtubules are assembled. The pathway leading to correctly folded alpha- and beta-tubulins is unusually complex: it involves cycles of ATP-dependent interaction of newly synthesized tubulin subunits with cytosolic chaperonin, resulting in the production of quasi-native folding intermediates, which must then be acted upon by additional protein cofactors. These cofactors form a supercomplex containing both alpha- and beta-tubulin polypeptides, from which native heterodimer is released in a GTP-dependent reaction. Here, we discuss the current state of our understanding of the function of cytosolic chaperonin and cofactors in tubulin folding
— id: 78377, year: 1997, vol: 7, page: 479, stat: Journal Article,

Tubulin subunits exist in an activated conformational state generated and maintained by protein cofactors
Tian G; Lewis SA; Feierbach B; Stearns T; Rommelaere H; Ampe C; Cowan NJ
1997 Aug 25;138(4):821-832, Journal of cell biology
The production of native alpha/beta tubulin heterodimer in vitro depends on the action of cytosolic chaperonin and several protein cofactors. We previously showed that four such cofactors (termed A, C, D, and E) together with native tubulin act on beta-tubulin folding intermediates generated by the chaperonin to produce polymerizable tubulin heterodimers. However, this set of cofactors generates native heterodimers only very inefficiently from alpha-tubulin folding intermediates produced by the same chaperonin. Here we describe the isolation, characterization, and genetic analysis of a novel tubulin folding cofactor (cofactor B) that greatly enhances the efficiency of alpha-tubulin folding in vitro. This enabled an integrated study of alpha- and beta-tubulin folding: we find that the pathways leading to the formation of native alpha- and beta-tubulin converge in that the folding of the alpha subunit requires the participation of cofactor complexes containing the beta subunit and vice versa. We also show that sequestration of native alpha-or beta-tubulins by complex formation with cofactors results in the destabilization and decay of the remaining free subunit. These data demonstrate that tubulin folding cofactors function by placing and/or maintaining alpha-and beta-tubulin polypeptides in an activated conformational state required for the formation of native alpha/beta heterodimers, and imply that each subunit provides information necessary for the proper folding of the other
— id: 7270, year: 1997, vol: 138, page: 821, stat: Journal Article,

Chaperonin-mediated folding of actin and tubulin
Lewis SA; Tian G; Vainberg IE; Cowan NJ
1996 Jan;132(1-2):1-4, Journal of cell biology
— id: 6929, year: 1996, vol: 132, page: 1, stat: Journal Article,

Pathway leading to correctly folded beta-tubulin
Tian G; Huang Y; Rommelaere H; Vandekerckhove J; Ampe C; Cowan NJ
1996 Jul 26;86(2):287-296, Cell
We describe the complete beta-tubulin folding pathway. Folding intermediates produced via ATP-dependent interaction with cytosolic chaperonin undergo a sequence of interactions with four proteins (cofactors A, D, E, and C). The postchaperonin steps in the reaction cascade do not depend on ATP or GTP hydrolysis, although GTP plays a structural role in tubulin folding. Cofactors A and D function by capturing and stabilizing beta-tubulin in a quasi-native conformation. Cofactor E binds to the cofactor D-beta-tubulin complex; interaction with cofactor C then causes the release of beta-tubulin polypeptides that are committed to the native state. Sequence analysis identifies yeast homologs of cofactors D (cin1) and E (pac2), characterized by mutations that affect microtubule function
— id: 56896, year: 1996, vol: 86, page: 287, stat: Journal Article,

The DNA-binding domain of the hexameric arginine repressor
Grandori, R; Lavoie, T A; Pflumm, M; Tian, G; Niersbach, H; Maas, W K; Fairman, R; Carey, J
1995 Nov 24;254(2):150-162, Journal of molecular biology
The arginine repressor of Escherichia coli is a classical feedback regulator, signalling the availability of L-arginine inside the cell. It differs from most other bacterial repressors in functioning as a hexamer, but structural details have been lacking and its shares no clear sequence homologies with other transcriptional regulators. Analysis of the amino acid residue sequence and proteolytic cleavage pattern of the repressor was used to identify a region predicted to house the DNA-binding function. When this protein fragment is overexpressed from a clone of the corresponding gene fragment, it represses ornithine transcarbamylase levels in vivo, and binds to the operator DNA in vitro, both in an arginine-independent manner. Sedimentation equilibrium and gel filtration indicate that the purified protein fragment is a monomer in solution. The results thus define the domain organization of the repressor at low resolution, suggesting that the N and C-terminal portions of the polypeptide chain are separated by a structural and functional border that decouples hexamerization and arginine binding from DNA binding
— id: 77936, year: 1995, vol: 254, page: 150, stat: Journal Article,

Quasi-native chaperonin-bound intermediates in facilitated protein folding
Tian G; Vainberg IE; Tap WD; Lewis SA; Cowan NJ
1995 Oct 13;270(41):23910-23913, Journal of biological chemistry
Chaperonins are known to facilitate protein folding, but their mechanism of action is not well understood. The fact that target proteins are released from and rebind to different chaperonin molecules ('cycling') during a folding reaction suggests that chaperonins function by unfolding aberrantly folded molecules, allowing them multiple opportunities to reach the native state in bulk solution. Here we show that the cycling of alpha-tubulin by cytosolic chaperonin (c-cpn) can be uncoupled from the action of cofactors required to complete the folding reaction. This results in the accumulation of folding intermediates which are chaperonin-bound, stable, and quasi-native in that they bind GTP nonexchangeably. We present evidence that these intermediates can be generated without the target protein leaving c-cpn. These data show that, in contrast to prevailing models, target proteins can maintain, and possibly acquire, significant native-like structure while chaperonin-bound
— id: 6872, year: 1995, vol: 270, page: 23910, stat: Journal Article,

Specificity in chaperonin-mediated protein folding
Tian G; Vainberg IE; Tap WD; Lewis SA; Cowan NJ
1995 May 18;375(6528):250-253, Nature
Chaperonins are ubiquitous multisubunit toroidal complexes that aid protein folding in an ATP-dependent manner. Current models of folding by the bacterial chaperonin GroEL depict its role as unfolding and releasing molecules that have misfolded, so that they can return to a potentially productive folding pathway in solution. Accordingly, a given target polypeptide might require several cycles of binding and ATP-driven release from different chaperonin complexes before reaching the native state. Surprisingly, cycling of a target protein does not guarantee its folding, and we report here that unfolded beta-actin or alpha-tubulin both form tight complexes when presented to either GroEL or its mitochondrial homologue, and both undergo cycles of release and rebinding upon incubation with ATP, but no native protein is produced. We conclude that different chaperonins produce distinctive spectra of folding intermediates
— id: 56720, year: 1995, vol: 375, page: 250, stat: Journal Article,

Explanation for different types of regulation of arginine biosynthesis in Escherichia coli B and Escherichia coli K12 caused by a difference between their arginine repressors
Tian G; Lim D; Oppenheim JD; Maas WK
1994 Jan 7;235(1):221-230, Journal of molecular biology
In Escherichia coli K12, formation of the enzymes of arginine biosynthesis are controlled by arginine, with complete repression during growth with added arginine, severe repression (about 95%) during growth without added arginine and complete derepression during arginine-limited growth. In E. coli B, the degree of repression is not correlated with arginine concentrations. Under all conditions of growth enzyme formation is repressed, with repression being somewhat less in a medium with arginine than in a medium without arginine. These differences in repressibility between the two strains have been shown previously to be due to the presence of different alleles of argR, the gene for the arginine repressor. Here we have compared the binding of the two repressors to the operator sites of argF (ARG boxes). In DNase I footprinting and gel retardation experiments with argF ARG boxes we have shown that the arginine repressor of E. coli K12 bound to arginine (ArgRK-arg) has a greater affinity than the arginine repressor of E. coli B bound to arginine (ArgRB-arg), whereas free ArgRB (ArgRBf) has a much stronger affinity than free ArgRK (ArgRKf). The stronger binding of ArgRBf can explain the repression seen in E. coli B during arginine-limited growth and indicates that ArgRBf, but not ArgRKf, is able to repress enzyme synthesis under physiological conditions. The weaker repression of E. coli B than of E. coli K12 seen in the presence of arginine can be explained by the lower affinity of ArgRB-arg for operator sites as compared to ArgRK-arg. Another contributing cause for the weaker repression is the reduction of ArgRBf concentration due to autoregulation of the gene for the repressor. Thus the combined effects of repression by ArgRBf, but not ArgRKf, with the weaker repression by ArgRB-arg as compared to ArgRK-arg, convert the arginine dependent regulation in E. coli K12 to arginine independent regulation in E. coli B
— id: 6537, year: 1994, vol: 235, page: 221, stat: Journal Article,

Mutational analysis of the arginine repressor of Escherichia coli
Tian, G; Maas, W K
1994 Aug;13(4):599-608, Molecular microbiology
Arginine biosynthesis in Escherichia coli is negatively regulated by a hexameric repressor protein, encoded by the gene argR and the corepressor arginine. By hydroxylamine mutagenesis two types of argR mutants were isolated and mapped. The first type is transdominant. In heterodiploids, these mutant polypeptides reduce the activity of the wild-type repressor, presumably by forming heteropolymers. Four mutant repressor proteins were purified. Two of these map in the N-terminal half of the protein. Gel retardation experiments showed that they bind poorly to DNA, but they could be precipitated by L-arginine at the same concentration as the wild-type repressor. The other two mutant repressors map in the C-terminal half of the protein. They are poorly precipitated by L-arginine and they bind poorly to DNA. In addition, one of these mutants appears to exist as a dimer. The second type of argR mutant repressor consists of super-repressors. Such mutants behave as arginine auxotrophs as a result of hyper-repression of arginine biosynthetic enzymes. They map at many locations throughout the argR gene. Three arginine super-repressor proteins were purified. In comparison with the wild-type repressor, two of them were shown to have a higher DNA-binding affinity in the absence of bound arginine, while the third was shown to have a higher DNA-binding affinity when bound to arginine
— id: 77939, year: 1994, vol: 13, page: 599, stat: Journal Article,

Binding of the arginine repressor of Escherichia coli K12 to its operator sites
Tian, G; Lim, D; Carey, J; Maas, W K
1992 Jul 20;226(2):387-397, Journal of molecular biology
In the arginine regulon of Escherichia coli K12 each of the eight operator sites consists of two 18-base-pair-long palindromic sequences called ARG boxes. In the operator sites for the structural genes of the regulon the two ARG boxes are separated by three base-pairs, in the regulatory gene argR they are separated by two base-pairs. The hexameric arginine repressor, the product of argR, binds to the two ARG boxes in an operator in the presence of L-arginine. From the results of various kinds of in vitro footprinting experiments with the ARG boxes of argF and argR (DNase I protection, hydroxyl radical, ethylation and methylation interference, methylation protection) it can be concluded that: (1) the repressor binds simultaneously to two adjacent ARG boxes; (2) that it binds on one face of the double helix; and (3) that it forms contacts with the major and minor grooves of each ARG box, but not with the central three base-pairs. The repressor can bind also to a single ARG box, but its affinity is about 100-fold lower than for two ARG boxes. From gel retardation experiments with 3H-labeled repressor and 32P-labeled argF operator DNA, it is concluded that the retarded DNA-protein complex contains no more than one repressor molecule per operator site and that most likely one hexamer binds to two ARG boxes. The bound repressor was shown to induce bending of argF operator DNA. The bending angle calculated from the results of gel retardation experiments is about 70 degrees and the bending center was located within the region encompassing the ARG boxes. The main features that distinguish the arginine repressor from other repressors studied in E. coli are its hexameric nature and the simultaneous binding of one hexameric molecule to two palindromic ARG boxes that are close to each other
— id: 77940, year: 1992, vol: 226, page: 387, stat: Journal Article,