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1.  Repressor of temperate mycobacteriophage L1 harbors a stable C-terminal domain and binds to different asymmetric operator DNAs with variable affinity 
Virology Journal  2007;4:64.
Background
Lysogenic mode of life cycle of a temperate bacteriophage is generally maintained by a protein called 'repressor'. Repressor proteins of temperate lambdoid phages bind to a few symmetric operator DNAs in order to regulate their gene expression. In contrast, repressor molecules of temperate mycobacteriophages and some other phages bind to multiple asymmetric operator DNAs. Very little is known at present about the structure-function relationship of any mycobacteriophage repressor.
Results
Using highly purified repressor (CI) of temperate mycobacteriophage L1, we have demonstrated here that L1 CI harbors an N-terminal domain (NTD) and a C-terminal domain (CTD) which are separated by a small hinge region. Interestingly, CTD is more compact than NTD at 25°C. Both CTD and CI contain significant amount of α-helix at 30°C but unfold partly at 42°C. At nearly 200 nM concentration, both proteins form appreciable amount of dimers in solution. Additional studies reveal that CI binds to O64 and OL types of asymmetric operators of L1 with variable affinity at 25°C. Interestingly, repressor – operator interaction is affected drastically at 42°C. The conformational change of CI is most possibly responsible for its reduced operator binding affinity at 42°C.
Conclusion
Repressors encoded by mycobacteriophages differ significantly from the repressor proteins of λ and related phages at functional level but at structural level they are nearly similar.
doi:10.1186/1743-422X-4-64
PMCID: PMC1934351  PMID: 17598887
2.  Structural analysis of the carboxy terminus of bacteriophage lambda repressor determined by antipeptide antibodies. 
Journal of Bacteriology  1989;171(3):1235-1244.
To analyze lambda repressor function and structure, antibodies were generated with synthetic peptides corresponding to sequences believed to be involved in prophage induction. These site-directed antibodies seemed to recognize preferentially the primary sequence of repressor because they reacted better in competition experiments with the oligopeptide and with the partially denatured forms of repressor than with the native molecules. This information, together with the characteristic ability of the antibodies to immunoprecipitate or react with repressor in immunoblots, allowed us to infer some conformational properties of the specific regions that the antibodies recognized. The antibodies reacted less with some mutant repressors that had a single amino acid substitution within the cognitive sequences. RecA-catalyzed cleavage of repressor was inhibited to different extents in relation to the proportion of repressor that each antipeptide immunoglobulin G (IgG) was able to immunoprecipitate. The antipeptide IgGs did not affect specific binding of repressor to operator DNA, whereas the antirepressor IgG was inhibitory. The three different IgGs competed for binding to repressor in an enzyme-linked immunosorbent assay additivity test, which suggested that the three regions of conserved amino acids are probably located on the same side of the carboxyl domain of repressor and possibly close together in the tertiary structure.
Images
PMCID: PMC209736  PMID: 2522089
3.  The N-Terminal Domain of the Repressor of Staphylococcus aureus Phage Φ11 Possesses an Unusual Dimerization Ability and DNA Binding Affinity 
PLoS ONE  2014;9(4):e95012.
Bacteriophage Φ11 uses Staphylococcus aureus as its host and, like lambdoid phages, harbors three homologous operators in between its two divergently oriented repressor genes. None of the repressors of Φ11, however, showed binding to all three operators, even at high concentrations. To understand why the DNA binding mechanism of Φ11 repressors does not match that of lambdoid phage repressors, we studied the N-terminal domain of the Φ11 lysogenic repressor, as it harbors a putative helix-turn-helix motif. Our data revealed that the secondary and tertiary structures of the N-terminal domain were different from those of the full-length repressor. Nonetheless, the N-terminal domain was able to dimerize and bind to the operators similar to the intact repressor. In addition, the operator base specificity, binding stoichiometry, and binding mechanism of this domain were nearly identical to those of the whole repressor. The binding affinities of the repressor and its N-terminal domain were reduced to a similar extent when the temperature was increased to 42°C. Both proteins also adequately dislodged a RNA polymerase from a Φ11 DNA fragment carrying two operators and a promoter. Unlike the intact repressor, the binding of the N-terminal domain to two adjacent operator sites was not cooperative in nature. Taken together, we suggest that the dimerization and DNA binding abilities of the N-terminal domain of the Φ11 repressor are distinct from those of the DNA binding domains of other phage repressors.
doi:10.1371/journal.pone.0095012
PMCID: PMC3991615  PMID: 24747758
4.  Dimerization specificity of P22 and 434 repressors is determined by multiple polypeptide segments. 
Journal of Bacteriology  1997;179(4):1253-1261.
The repressor protein of bacteriophage P22 binds to DNA as a homodimer. This dimerization is absolutely required for DNA binding. Dimerization is mediated by interactions between amino acids in the carboxyl (C)-terminal domain. We have constructed a plasmid, p22CT-1, which directs the overproduction of just the C-terminal domain of the P22 repressor (P22CT-1). Addition of P22CT-1 to DNA-bound P22 repressor causes the dissociation of the complex. Cross-linking experiments show that P22CT-1 forms specific heterodimers with the intact P22 repressor protein, indicating that inhibition of P22 repressor DNA binding by P22CT-1 is mediated by the formation of DNA binding-inactive P22 repressor:P22CT-1 heterodimers. We have taken advantage of the highly conserved amino acid sequences within the C-terminal domains of the P22 and 434 repressors and have created chimeric proteins to help identify amino acid regions required for dimerization specificity. Our results indicate that the dimerization specificity region of these proteins is concentrated in three segments of amino acid sequence that are spread across the C-terminal domain of each of the two phage repressors. We also show that the set of amino acids that forms the cooperativity interface of the P22 repressor may be distinct from those that form its dimer interface. Furthermore, cooperativity studies of the wild-type and chimeric proteins suggest that the location of cooperativity interface in the 434 repressor may also be distinct from that of its dimerization interface. Interestingly, changes in the dimer interface decreases the ability of the 434 repressor to discriminate between its wild-type binding sites, O(R)1, O(R)2, and O(R)3. Since 434 repressor discrimination between these sites depends in large part on the ability of this protein to recognize sequence-specific differences in DNA structure and flexibility, this result indicates that the C-terminal domain is intimately involved in the recognition of sequence-dependent differences in DNA structure and flexibility.
PMCID: PMC178823  PMID: 9023209
5.  The Bacteriophage 434 Repressor Dimer Preferentially Undergoes Autoproteolysis by an Intramolecular Mechanism 
Journal of Bacteriology  2005;187(16):5624-5630.
Inactivation of the lambdoid phage repressor protein is necessary to induce lytic growth of a lambdoid prophage. Activated RecA, the mediator of the host SOS response to DNA damage, causes inactivation of the repressor by stimulating the repressor's nascent autocleavage activity. The repressor of bacteriophage lambda and its homolog, LexA, preferentially undergo RecA-stimulated autocleavage as free monomers, which requires that each monomer mediates its own (intramolecular) cleavage. The cI repressor of bacteriophage 434 preferentially undergoes autocleavage as a dimer specifically bound to DNA, opening the possibility that one 434 repressor subunit may catalyze proteolysis of its partner subunit (intermolecular cleavage) in the DNA-bound dimer. Here, we first identified and mutagenized the residues at the cleavage and active sites of 434 repressor. We utilized the mutant repressors to show that the DNA-bound 434 repressor dimer overwhelmingly prefers to use an intramolecular mechanism of autocleavage. Our data suggest that the 434 repressor cannot be forced to use an intermolecular cleavage mechanism. Based on these data, we propose a model in which the cleavage-competent conformation of the repressor is stabilized by operator binding.
doi:10.1128/JB.187.16.5624-5630.2005
PMCID: PMC1196080  PMID: 16077107
6.  Dimerization of the Agrobacterium tumefaciens VirB4 ATPase and the effect of ATP-binding cassette mutations on the assembly and function of the T-DNA transporter 
Molecular microbiology  1999;32(6):1239-1253.
Summary
The Agrobacterium tumefaciens VirB4 ATPase functions with other VirB proteins to export T-DNA to susceptible plant cells and other DNA substrates to a variety of prokaryotic and eukaryotic cells. Previous studies have demonstrated that VirB4 mutants with defects in the Walker A nucleotide-binding motif are non-functional and exert a dominant negative phenotype when synthesized in wild-type cells. This study characterized the oligomeric structure of VirB4 and examined the effects of Walker A sequence mutations on complex formation and transporter activity. VirB4 directed dimer formation when fused to the amino-terminal portion of cI repressor protein, as shown by immunity of Escherichia coli cells to λ phage infection. VirB4 also dimerized in Agrobacterium tumefaciens, as demonstrated by the recovery of a detergent-resistant complex of native protein and a functional, histidine-tagged derivative by precipitation with anti-His6 antibodies and by Co2+ affinity chromatography. Walker A sequence mutants directed repressor dimerization in E. coli and interacted with His-VirB4 in A. tumefaciens, indicating that ATP binding is not required for self-association. A dimerization domain was localized to a proposed N-terminal membrane-spanning region of VirB4, as shown by the dominance of an allele coding for the N-terminal 312 residues and phage immunity of host cells expressing cI repressor fusions to alleles for the first 237 or 312 residues. A recent study reported that the synthesis of a subset of VirB proteins, including VirB4, in agrobacterial recipients has a pronounced stimulatory effect on the virB-dependent conjugal transfer of plasmid RSF1010 by agrobacterial donors. VirB4′312 suppressed the stimulatory effect of VirB proteins for DNA uptake when synthesized in recipient cells. In striking contrast, Walker A sequence mutants contributed to the stimulatory effect of VirB proteins to the same extent as native VirB4. These findings indicate that the oligomeric structure of VirB4, but not its capacity to bind ATP, is important for the assembly of VirB proteins as a DNA uptake system. The results of these studies support a model in which VirB4 dimers or homomultimers contribute structural information for the assembly of a transenvelope channel competent for bidirectional DNA transfer, whereas an ATP-dependent activity is required for configuring this channel as a dedicated export machine.
PMCID: PMC3918219  PMID: 10383764
7.  The Molecular Mechanisms of Allosteric Mutations Impairing MepR Repressor Function in Multidrug-Resistant Strains of Staphylococcus aureus 
mBio  2013;4(5):e00528-13.
ABSTRACT
Overexpression of the Staphylococcus aureus multidrug efflux pump MepA confers resistance to a wide variety of antimicrobials. mepA expression is controlled by MarR family member MepR, which represses mepA and autorepresses its own production. Mutations in mepR are a primary cause of mepA overexpression in clinical isolates of multidrug-resistant S. aureus. Here, we report crystal structures of three multidrug-resistant MepR variants, which contain the single-amino-acid substitution A103V, F27L, or Q18P, and wild-type MepR in its DNA-bound conformation. Although each mutation impairs MepR function by decreasing its DNA binding affinity, none is located in the DNA binding domain. Rather, all are found in the linker region connecting the dimerization and DNA binding domains. Specifically, the A103V substitution impinges on F27, which resolves potential steric clashes via displacement of the DNA binding winged-helix-turn-helix motifs that lead to a 27-fold reduction in DNA binding affinity. The F27L substitution forces F104 into an alternative rotamer, which kinks helix 5, thereby interfering with the positioning of the DNA binding domains and decreasing mepR operator affinity by 35-fold. The Q18P mutation affects the MepR structure and function most significantly by either creating kinks in the middle of helix 1 or completely unfolding its C terminus. In addition, helix 5 of Q18P is either bent or completely dissected into two smaller helices. Consequently, DNA binding is diminished by 2,000-fold. Our structural studies reveal heretofore-unobserved allosteric mechanisms that affect repressor function of a MarR family member and result in multidrug-resistant Staphylococcus aureus.
IMPORTANCE
Staphylococcus aureus is a major health threat to immunocompromised patients. S. aureus multidrug-resistant variants that overexpress the multidrug efflux pump mepA emerge frequently due to point mutations in MarR family member MepR, the mepA transcription repressor. Significantly, the majority of MepR mutations identified in these S. aureus clinical isolates are found not in the DNA binding domain but rather in a linker region, connecting the dimerization and DNA binding domains. The location of these mutants underscores the critical importance of a properly functioning allosteric mechanism that regulates MepR function. Understanding the dysregulation of such allosteric MepR mutants underlies this study. The high-resolution structures of three such allosteric MepR mutants reveal unpredictable conformational consequences, all of which preclude cognate DNA binding, while biochemical studies emphasize their debilitating effects on DNA binding affinity. Hence, mutations in the linker region of MepR and their structural consequences are key generators of multidrug-resistant Staphylococcus aureus.
doi:10.1128/mBio.00528-13
PMCID: PMC3760248  PMID: 23982071
8.  RelB and RelE of Escherichia coli Form a Tight Complex That Represses Transcription via the Ribbon–Helix–Helix Motif in RelB 
Journal of Molecular Biology  2009;394(2):183-196.
RelB, the ribbon–helix–helix (RHH) repressor encoded by the relBE toxin–antitoxin locus of Escherichia coli, interacts with RelE and thereby counteracts the mRNA cleavage activity of RelE. In addition, RelB dimers repress the strong relBE promoter and this repression by RelB is enhanced by RelE; that is, RelE functions as a transcriptional co-repressor. RelB is a Lon protease substrate, and Lon is required both for activation of relBE transcription and for activation of the mRNA cleavage activity of RelE. Here we characterize the molecular interactions important for transcriptional control of the relBE model operon. Using an in vivo screen for relB mutants, we identified multiple nucleotide changes that map to important amino acid positions within the DNA-binding domain formed by the N-terminal RHH motif of RelB. Analysis of DNA binding of a subset of these mutant RHH proteins by gel-shift assays, transcriptional fusion assays and a structure model of RelB–DNA revealed amino acid residues making crucial DNA–backbone contacts within the operator (relO) DNA. Mutational and footprinting analyses of relO showed that RelB dimers bind on the same face of the DNA helix and that the RHH motif recognizes four 6-bp repeats within the bipartite binding site. The spacing between each half-site was found to be essential for cooperative interactions between adjacently bound RelB dimers stabilized by the co-repressor RelE. Kinetic and stoichiometric measurements of the interaction between RelB and RelE confirmed that the proteins form a high-affinity complex with a 2:1 stoichiometry. Lon degraded RelB in vitro and degradation was inhibited by RelE, consistent with the proposal that RelE protects RelB from proteolysis by Lon in vivo.
doi:10.1016/j.jmb.2009.09.006
PMCID: PMC2812701  PMID: 19747491
RHH, ribbon–helix–helix; TA, toxin–antitoxin; WT, wild type; SPR, surface plasmon resonance; HMK, heart myosin kinase; X-gal, 5-bromo-4-chloro-3-indoyl-β-d-galactoside; EDTA, ethylenediaminetetraacetic acid; toxin; antitoxin; RelB; RelE; ribbon–helix–helix
9.  Mutations Partially Inactivating the Lactose Repressor of Escherichia coli 
Journal of Bacteriology  1974;119(2):500-507.
After treatment with N-methyl-N′-nitro-N-nitrosoguanidine, 133 independent mutants of a haploid strain of Escherichia coli able to use phenyl-β-galactoside as a carbon source were obtained. The galactoside was specific in selecting for mutants with increases in their uninduced levels of β-galactosidase. Virtually all mutants (37 in a subsample of 38) carried mutations in the lac repressor gene. There were two classes of repressor mutants. As well as the commonly identified class of mutants with completely inactivated repressors, there was a frequent class of mutants (21/37) whose repressors were partially inactivated. Most of these (15/21) repressed β-galactosidase synthesis 4 to 50 times less than wild type, but were more numerous in the lower part of this range. Their β-galactosidase was inducible to levels characteristic of the parent strain. The repressor activities were diverse and stably expressed under routine growth conditions. The decreased activity did not result from the formation of temperature-sensitive repressors. None of the mutants with completely inactivated repressors appeared to carry UAG or UGA chain-terminating codons. On the assumption that the partially defective repressor mutants carried missense mutations, it is argued that missense mutations in the lac repressor gene modify the repressor's affinity for the operator with high probability. An explanation is proposed for the apparent sensitivity of this repressor function to partial inactivation as the result of amino acid substitutions.
PMCID: PMC245633  PMID: 4604800
10.  Amino acid substitutions in the CytR repressor which alter its capacity to regulate gene expression. 
Journal of Bacteriology  1992;174(9):2881-2890.
In Escherichia coli, transport and catabolism of nucleosides require expression of the genes composing the CytR regulon. Transcription initiation of cistrons in this gene family is activated by cyclic AMP-catabolite activator protein (cAMP-CAP), repressed by the CytR protein, and induced by cytidine. A random proofreading mutagenesis procedure and a genetic screen using udp-lac fusions have allowed the identification of distinct regions of the 341-amino-acid CytR polypeptide that are critical for repression of gene expression and response to induction. Determination of the ability of various CytR mutants to control gene expression in vivo indicated that the intrinsic affinity of the CytR protein for operator DNA is gene specific and that efficient repression of transcription by wild-type CytR is dependent on the interaction of CytR with cAMP-CAP. CytR mutants that were cytidine induction defective (CID) were characterized; these mutant proteins had only Asp-281 replaced. Data obtained with cytR delta M149, a dominant negative allele, indicated that the native CytR repressor is an oligomeric protein. Representative cytR mutations were combined with cytR delta M149, and the resulting hybrid repressors were tested for transdominance in a CytR+ E. coli strain. Amino acid substitutions A209E and C289Y suppressed the transdominance of CytR delta M149, suggesting that these replacements alter the normal protein contacts involved in repressor subunit-subunit association. In contrast, amino acid substitutions located in the N-terminal portion of the CytR protein had no effect on the transdominance of CytR delta M149. The results from this study suggest that the CytR repressor is an oligomeric, allosteric protein in which conformational changes are required for repression and derepression.
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PMCID: PMC205940  PMID: 1569019
11.  An Unusual Phage Repressor Encoded by Mycobacteriophage BPs 
PLoS ONE  2015;10(9):e0137187.
Temperate bacteriophages express transcription repressors that maintain lysogeny by down-regulating lytic promoters and confer superinfection immunity. Repressor regulation is critical to the outcome of infection—lysogenic or lytic growth—as well as prophage induction into lytic replication. Mycobacteriophage BPs and its relatives use an unusual integration-dependent immunity system in which the phage attachment site (attP) is located within the repressor gene (33) such that site-specific integration leads to synthesis of a prophage-encoded product (gp33103) that is 33 residues shorter at its C-terminus than the virally-encoded protein (gp33136). However, the shorter form of the repressor (gp33103) is stable and active in repression of the early lytic promoter PR, whereas the longer virally-encoded form (gp33136) is inactive due to targeted degradation via a C-terminal ssrA-like tag. We show here that both forms of the repressor bind similarly to the 33–34 intergenic regulatory region, and that BPs gp33103 is a tetramer in solution. The BPs gp33103 repressor binds to five regulatory regions spanning the BPs genome, and regulates four promoters including the early lytic promoter, PR. BPs gp33103 has a complex pattern of DNA recognition in which a full operator binding site contains two half sites separated by a variable spacer, and BPs gp33103 induces a DNA bend at the full operator site but not a half site. The operator site structure is unusual in that one half site corresponds to a 12 bp palindrome identified previously, but the other half site is a highly variable variant of the palindrome.
doi:10.1371/journal.pone.0137187
PMCID: PMC4557955  PMID: 26332854
12.  Control of translational repression by protein-protein interactions. 
Nucleic Acids Research  1992;20(7):1649-1655.
The coat protein of the RNA bacteriophage MS2 is a translational repressor and interacts with a specific RNA stem-loop to inhibit translation of the viral replicase gene. As part of an effort to dissect genetically its RNA binding function, mutations were identified in the coat protein sequence that suppress mutational defects in the translational operator. Each of the mutants displayed a super-repressor phenotype, repressing translation from the wild-type and a variety of mutant operators better than did the wild-type coat protein. At least one mutant probably binds RNA more tightly than wild-type. The other mutants, however, were defective for assembly of virus-like particles, and self-associated predominantly as dimers. It is proposed that this assembly defect accounts for their super-repressor characteristics, since failure to assemble into virus-like particles elevates the effective concentration of repressor dimers. This hypothesis is supported by the observation that deletion of thirteen amino acids known to be important for assembly of dimers into capsids also resulted in the same assembly defect and in super-repressor activity. A second class of assembly defects is also described. Deletion of two amino acids from the C-terminus of coat protein resulted in failure to form capsids, most of the coat protein having the apparent molecular weight expected of trimers. This mutant (dl-8) was completely defective for repressor activity, probably because of an inability to form dimers. These results point out the inter-dependence of the structural and regulatory functions of coat protein.
Images
PMCID: PMC312251  PMID: 1579455
13.  The NH2-terminal arms of trp repressor participate in repressor/operator association. 
Nucleic Acids Research  1992;20(2):337-341.
The 3-dimensional structure of the trp repressor, aporepressor, and repressor/operator complex have been described. The NH2-terminal arms of the protein, comprising approximately 12-14 residues, were not well resolved in any of these structures. Previous studies by Carey showed that the arms are required for full in vitro repressor activity. To examine the roles of the arms more fully we have removed codons 2-5 and 2-8 of the trpR gene and analyzed the resulting truncated repressors in vivo and in vitro. The delta 2-5 trp repressor was found to be approximately 25% as active as the wild type repressor in vivo. In in vitro equilibrium binding experiments, the delta 2-5 trp repressor was shown to be five-fold less active in operator binding. The rate of dissociation of the complex formed between the delta 2-5 trp repressor and operator was essentially the same as the rate of dissociation of the wild type trp repressor/operator complex. However association of the delta 2-5 trp repressor with operator was clearly defective. Since the NH2-terminal arms of the trp repressor appear to affect association predominantly they may play a role in facilitating non-specific association of repressor with DNA as repressor seeks its cognate operators. The delta 2-8 trp repressor was unstable in vivo and in vitro, suggesting that some portion of the NH2-terminal arm is required for proper folding of the remainder of the molecule.
PMCID: PMC310375  PMID: 1741259
14.  Expression, purification, and functional characterization of the carboxyl-terminal domain fragment of bacteriophage 434 repressor. 
Journal of Bacteriology  1994;176(22):6907-6914.
The repressor protein of bacteriophage 434 binds to DNA as a dimer of identical subunits. Its strong dimerization is mediated by the carboxyl-terminal domain. Cooperative interactions between the C-terminal domains of two repressor dimers bound at adjacent sites can stabilize protein-DNA complexes formed with low-affinity binding sites. We have constructed a plasmid, pCT1, which directs the overproduction of the carboxyl-terminal domain of 434 repressor. The protein encoded by this plasmid is called CT-1. Cells transformed with pCT1 are unable to be lysogenized by wild-type 434 phage, whereas control cells are lysogenized at an efficiency of 1 to 5%. The CT-1-mediated interference with lysogen formation presumably results from formation of heteromeric complexes between the phage-encoded repressor and the plasmid-encoded carboxyl-terminal domain fragment. These heteromers are unable to bind DNA and thereby inhibit the repressor's activity in promoting lysogen formation. Two lines of evidence support this conclusion. First, DNase I footprinting experiments show that at a 2:1 ratio of CT-1 to intact 434 repressor, purified CT-1 protein prevents the formation of complexes between 434 repressor and its OR1 binding site. Second, cross-linking experiments reveal that only a specific heterodimeric complex forms between CT-1 and intact 434 repressor. This latter observation indicates that CT-1 interferes with 434 repressor-operator complex formation by preventing dimerization and not by altering the conformation of the DNA-bound repressor dimer. Our other evidence is also consistent with this suggestion. We have used deletion analysis in an attempt to define the region which mediates the 434 repressor-CT-1 interaction. CT-1 proteins which have more than the last 14 amino acids removed are unable to interfere with 434 repressor action in vivo.
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PMCID: PMC197060  PMID: 7961451
15.  Interconvertible Lac Repressor–DNA Loops Revealed by Single-Molecule Experiments 
PLoS Biology  2008;6(9):e232.
At many promoters, transcription is regulated by simultaneous binding of a protein to multiple sites on DNA, but the structures and dynamics of such transcription factor-mediated DNA loops are poorly understood. We directly examined in vitro loop formation mediated by Escherichia coli lactose repressor using single-molecule structural and kinetics methods. Small (∼150 bp) loops form quickly and stably, even with out-of-phase operator spacings. Unexpectedly, repeated spontaneous transitions between two distinct loop structures were observed in individual protein–DNA complexes. The results imply a dynamic equilibrium between a novel loop structure with the repressor in its crystallographic “V” conformation and a second structure with a more extended linear repressor conformation that substantially lessens the DNA bending strain. The ability to switch between different loop structures may help to explain how robust transcription regulation is maintained even though the mechanical work required to form a loop may change substantially with metabolic conditions.
Author Summary
Some proteins that regulate DNA transcription do so by binding simultaneously to two separated sites on the DNA molecule, forming a DNA loop. Although such loops are common, many of their features are poorly characterized. Of particular interest is the question of how some proteins accommodate the formation of loops of different sizes, particularly when the loops are small and thus require strong bending (and, in some cases, twisting) of the DNA to form. We observed the shape and behavior of individual DNA molecules bent into tight loops by Lac repressor, a transcription-regulating protein from the bacterium Escherichia coli. Loops were formed in DNA molecules with repressor-binding sites on opposite faces of the DNA double helix almost as readily as in those with sites on the same side, suggesting that the repressor is highly flexible. The DNA can switch back and forth between a tighter and a looser loop structure “on the fly” during the lifetime of a single loop, further evidence that Lac repressor is capable of adopting different shapes that may serve to minimize DNA bending or twisting in loops. The ability of the repressor to readily switch between different loop shapes may allow it to maintain effective control of transcription across situations in which the difficulty of bending or twisting DNA changes substantially.
A large-scale conformational change in a transcription factor protein allows DNA loops to dynamically switch between alternative conformations that may contribute to robust transcription regulation.
doi:10.1371/journal.pbio.0060232
PMCID: PMC2553838  PMID: 18828671
16.  Internal Dynamics of the Tryptophan Repressor (TrpR) and Two Functionally Distinct TrpR Variants, L75F-TrpR and A77V-TrpR, in their L-Trp bound forms 
Biochemistry  2011;50(23):5140-5153.
Backbone amide dynamics of the Escherichia coli tryptophan repressor protein (WT-TrpR) and two functionally distinct variants, L75F-TrpR and A77V-TrpR, in their holo-(L-tryptophan (L-Trp) co-repressor-bound) form have been characterized using 15N NMR relaxation. The three proteins possess very similar structures ruling out major conformational differences as the source of their functional differences, and suggest that changes in protein flexibility are at the origin of their distinct functional properties. Comparison of site specific 15N-T1, 15N-T2, 15N-{1H}-nOes, reduced spectral density, and generalized order parameters (S2), indicates that backbone dynamics in the three holo-repressors are overall very similar with a few notable and significant exceptions for backbone atoms residing within the proteins’ DNA-binding domain. We find that flexibility is highly restricted for amides in core α-helices (i.e. helices A, B, C, and F), and a comparable “stiffening” is observed for residues in the DNA recognition helix (helix E) of the helix-D-turn-helix-E (HTH) DNA-binding domain of the three holo-repressors. Unexpectedly, amides located in helix D and in adjacent turn regions remain flexible. These data support the concept that residual flexibility in TrpR is essential for repressor function, DNA-binding, and molecular recognition of target operators. Comparison of the 15N NMR relaxation parameters of the holo-TrpRs with those of the apo-TrpRs indicate that the single point amino acid substitutions, L75F and A77V, perturb the flexibility of backbone amides of TrpR in very different ways, and are most pronounced in the apo-forms of the three repressors. Finally, we present these findings in the context of other DNA-binding proteins and the role of protein flexibility in molecular recognition.
doi:10.1021/bi200389k
PMCID: PMC3113449  PMID: 21553830
Tryptophan; repressor; protein dynamics; NMR relaxation
17.  Functional domains of the penicillinase repressor of Bacillus licheniformis. 
Journal of Bacteriology  1993;175(22):7383-7390.
The penicillinase repressor (PENI) negatively regulates expression of the penicillinase gene (penP) in Bacillus licheniformis by binding to its operators located within the promoter region of penP.penI codes for a protein with 128 amino acids. Filter-binding analyses suggest that the active form of the repressor is a dimer. Genetic analyses of PENI derivatives showed that the repressor carrying either a 6-amino-acid deletion near the N terminus or a 14-amino-acid deletion at the C terminus was functionally inactive in vivo. A repressor derivative carrying a 6-amino-acid deletion within its N-terminal region was extensively purified and used in DNA footprinting and subunit cross-linking analyses. The results of these studies showed that the repressor derivative had lost its ability to bind operator specifically even though it could dimerize effectively. In similar studies, we demonstrated that an N-terminal portion of PENI with a molecular mass of 10 kDa derived by digestion with papain was able to bind operator specifically but with reduced affinity and had completely lost its ability to dimerize. These data suggest that the repressor has two functional and separable domains. The amino-terminal domain of the repressor is responsible for operator recognition, and the carboxyl-terminal domain is involved in subunit dimerization.
Images
PMCID: PMC206883  PMID: 8226686
18.  Cd(II)-Responsive and Constitutive Mutants Implicate a Novel Domain in MerR 
Journal of Bacteriology  1999;181(11):3462-3471.
Expression of the Tn21 mercury resistance (mer) operon is controlled by a metal-sensing repressor-activator, MerR. When present, MerR always binds to the same position on the DNA (the operator merO), repressing transcription of the structural genes merTPCAD in the absence of Hg(II) and inducing their transcription in the presence of Hg(II). Although it has two potential binding sites, the purified MerR homodimer binds only one Hg(II) ion, employing Cys82 from one monomer and Cys117 and Cys126 from the other. When MerR binds Hg(II), it changes allosterically and also distorts the merO DNA to facilitate transcriptional initiation by ς70 RNA polymerase. Wild-type MerR is highly specific for Hg(II) and is 100- and 1,000-fold less responsive to the chemically related group 12 metals, Cd(II) and Zn(II), respectively. We sought merR mutants that respond to Cd(II) and obtained 11 Cd(II)-responsive and 5 constitutive mutants. The Cd(II)-responsive mutants, most of which had only single-residue replacements, were also repression deficient and still Hg(II) responsive but, like the wild type, were completely unresponsive to Zn(II). None of the Cd(II)-responsive mutations occurred in the DNA binding domain or replaced any of the key Cys residues. Five Cd(II)-responsive single mutations lie in the antiparallel coiled-coil domain between Cys82 and Cys117 which constitutes the dimer interface. These mutations identify 10 new positions whose alteration significantly affect MerR’s metal responsiveness or its repressor function. They give rise to specific predictions for how MerR distinguishes group 12 metals, and they refine our model of the novel domain structure of MerR. Secondary-structure predictions suggest that certain elements of this model also apply to other MerR family regulators.
PMCID: PMC93814  PMID: 10348859
19.  The metalloregulatory zinc site in Streptococcus pneumoniae AdcR, a zinc-activated MarR-family repressor 
Journal of molecular biology  2010;403(2):197-216.
Streptococcus pneumoniae D39 AdcR (adhesin competence repressor) is the first metal-sensing member of the MarR (multiple antibiotic resistance repressor) family to be characterized. Expression profiling with a ΔadcR strain grown in liquid culture (brain heart infusion; BHI) under microaerobic conditions reveals upregulation of 13 genes including adcR and adcCBA, encoding a high affinity ABC uptake system for zinc, and genes encoding cell-surface zinc-binding pneumococcal histidine triad (Pht) proteins and AdcAII (Lmb, laminin binding). The ΔadcR, H108Q and H112Q adcR mutant allelic strains grown in 0.2 mM Zn(II) exhibit a slow-growth phenotype and a ≈2-fold increase in cell-associated Zn(II). Apo- and Zn(II)-bound AdcR are homodimers in solution and binding to a 28-mer DNA containing an adc operator is strongly stimulated by Zn(II) with KDNA-Zn = 2.4 ×108 M−1 (pH 6.0, 0.2 M NaCl, 25 °C). AdcR binds two Zn(II) per dimer, with step-wise Zn(II) affinities KZn1 and KZn2 of ≥109 M−1 at pH 6.0 and ≥1012 M−1 at pH 8.0. X-ray absorption spectroscopy (XAS) of the high affinity site reveals a pentacoordinate N/O complex and no cysteine coordination, the latter finding corroborated by wild-type-like functional properties of C30A AdcR. Alanine substitution of conserved residues His42 in the DNA binding domain, and His108 and His112 in the C-terminal regulatory domain, abolish high affinity Zn(II) binding and greatly reduce Zn(II)-activated binding to DNA. NMR studies reveal that these mutants adopt the same folded conformation as dimeric wild-type apo AdcR, but fail to conformationally switch upon Zn(II) binding. These studies clearly identify His42, His108 and H112 as metalloregulatory zinc ligands in S. pneumoniae AdcR.
doi:10.1016/j.jmb.2010.08.030
PMCID: PMC2949468  PMID: 20804771
20.  Crystal Structures of the BlaI Repressor from Staphylococcus aureus and Its Complex with DNA: Insights into Transcriptional Regulation of the bla and mec Operons 
Journal of Bacteriology  2005;187(5):1833-1844.
The 14-kDa BlaI protein represses the transcription of blaZ, the gene encoding β-lactamase. It is homologous to MecI, which regulates the expression of mecA, the gene encoding the penicillin binding protein PBP2a. These genes mediate resistance to β-lactam antibiotics in staphylococci. Both repressors can bind either bla or mec DNA promoter-operator sequences. Regulated resistance genes are activated via receptor-mediated cleavage of the repressors. Cleavage is induced when β-lactam antibiotics bind the extramembrane sensor of the sensor-transducer signaling molecules, BlaR1 or MecR1. The crystal structures of BlaI from Staphylococcus aureus, both in free form and in complex with 32 bp of DNA of the mec operator, have been determined to 2.0- and 2.7-Å resolutions, respectively. The structure of MecI, also in free form and in complex with the bla operator, has been previously reported. Both repressors form homodimers, with each monomer composed of an N-terminal DNA binding domain of winged helix-turn-helix topology and a C-terminal dimerization domain. The structure of BlaI in complex with the mec operator shows a protein-DNA interface that is conserved between both mec and bla targets. The recognition helix α3 interacts specifically with the conserved TACA/TGTA DNA binding motif. BlaI and, probably, MecI dimers bind to opposite faces of the mec DNA double helix in an up-and-down arrangement, whereas MecI and, probably, BlaI dimers bind to the same DNA face of bla promoter-operator DNA. This is due to the different spacing of mec and bla DNA binding sites. Furthermore, the flexibility of the dimeric proteins may make the C-terminal proteolytic cleavage site more accessible when the repressors are bound to DNA than when they are in solution, suggesting that the induction cascade involves bound rather than free repressor.
doi:10.1128/JB.187.5.1833-1844.2005
PMCID: PMC1064009  PMID: 15716455
21.  Functional domains of the human orphan receptor ARP-1/COUP-TFII involved in active repression and transrepression. 
Molecular and Cellular Biology  1997;17(9):4914-4932.
The orphan receptor ARP-1/COUP-TFII, a member of the chicken ovalbumin upstream promoter transcription factor (COUP-TF) subfamily of nuclear receptors, strongly represses transcriptional activity of numerous genes, including several apolipoprotein-encoding genes. Recently it has been demonstrated that the mechanism by which COUP-TFs reduce transcriptional activity involves active repression and transrepression. To map the domains of ARP-1/COUP-TFII required for repressor activity, a detailed deletion analysis of the protein was performed. Chimeric proteins in which various segments of the ARP-1/COUP-TFII carboxy terminus were fused to the GAL4 DNA binding domain were used to characterize its active repression domain. The smallest segment confering active repressor activity to a heterologous DNA binding domain was found to comprise residues 210 to 414. This domain encompasses the region of ARP-1/COUP-TFII corresponding to helices 3 to 12 in the recently published crystal structure of other members of the nuclear receptor superfamily. It includes the AF-2 AD core domain formed by helix 12 but not the hinge region, which is essential for interaction with a corepressor in the case of the thyroid hormone and retinoic acid receptor. Attachment of the nuclear localization signal from the simian virus 40 large T antigen (Flu tag) to the amino terminus of ARP-1/COUP-TFII abolished its ability to bind to DNA without affecting its repressor activity. By using a series of Flu-tagged mutants, the domains required for transrepressor activity of the protein were mapped. They include the DNA binding domain and the segment spanning residues 193 to 399. Transcriptional activity induced by liver-enriched transactivators such as hepatocyte nuclear factor 3 (HNF-3), C/EBP, or HNF-4 was repressed by ARP-1/COUP-TFII independent of the presence of its cognate binding site, while basal transcription or transcriptional activity induced by ATF or Sp1 was not perturbed by the protein. In conclusion, our results demonstrate that the domains of ARP-1/COUP-TFII required for active repression and transrepression do not coincide. Moreover, they strongly suggest that transrepression is the predominant mechanism underlying repressor activity of ARP-1/COUP-TFII. This mechanism most likely involves interaction of the protein with one or several transcriptional coactivator proteins which are employed by various liver-enriched transactivators but not by ubiquitous factors such as Sp1 or ATF.
PMCID: PMC232344  PMID: 9271371
22.  A Role for the PERIOD:PERIOD Homodimer in the Drosophila Circadian Clock 
PLoS Biology  2009;7(4):e1000003.
Circadian clocks in eukaryotes rely on transcriptional feedback loops, in which clock genes repress their own transcription resulting in molecular oscillations with a period of ∼24 h. In Drosophila, the clock proteins Period (PER) and Timeless (TIM) operate in such a feedback loop, whereby they first accumulate in the cytoplasm of clock cells as a heterodimer. Nuclear translocation of the complex or the individual PER and TIM proteins is followed by repression of per and tim transcription, whereby PER seems to act as the prime repressor. We found that in addition to PER:TIM complexes, functional PER:PER homodimers exist in flies. Specific disruption of PER homodimers results in drastically impaired behavioral and molecular rhythmicity, pointing the biological importance of this clock protein complex. Analysis of PER subcellular distribution and repressor competence in the PER dimer mutant revealed defects in PER nuclear translocation and a disruption of rhythmic period transcription. The striking similarity of these phenotypes with that of reduced CKII activity suggests that the formation or function of the PER dimer is closely linked to this kinase. Our results confirm a previous structural model for PER and provide strong evidence that PER homodimers are important for circadian clock function.
Author Summary
The current models of circadian clocks in flies and mammals involve the formation of complexes between clock proteins in the cytoplasm. These complexes are usually heterodimers (that is, made up of two different clock proteins) and appear to enter the nucleus at certain times of the circadian day in order to shut down their own gene expression by deactivating specific transcription factors. After progressive phosphorylation the repressor proteins eventually are degraded so that a new cycle of transcription can begin. Here we present evidence that in addition to heterodimeric complexes, the clock protein PERIOD (PER) also forms homodimers (pairs of identical proteins). Based on a structural model a PER mutant was designed, which is not able to form homodimers but can still bind to its partner TIMELESS (TIM). Flies expressing this mutant PER protein show abnormal clock function in regard to PER nuclear translocation, repressor activity, and behavioral rhythms. The circadian clock model in flies therefore needs to be extended by adding the PER:PER homodimer as a functional unit. Recent structural studies with mammalian PER proteins suggest that homodimers between clock proteins are an important general feature of eukaryotic clocks.
The circadian molecular clock model needs to be extended by adding the PERIOD:PERIOD homodimer as a functional unit in rhythm generation in Drosophila. Blocking this dimerization leads to faulty nuclear localization, reduced repressor activity, and impaired behavioral rhythms.
doi:10.1371/journal.pbio.1000003
PMCID: PMC2671555  PMID: 19402744
23.  The Transcription Factor AmrZ Utilizes Multiple DNA Binding Modes to Recognize Activator and Repressor Sequences of Pseudomonas aeruginosa Virulence Genes 
PLoS Pathogens  2012;8(4):e1002648.
AmrZ, a member of the Ribbon-Helix-Helix family of DNA binding proteins, functions as both a transcriptional activator and repressor of multiple genes encoding Pseudomonas aeruginosa virulence factors. The expression of these virulence factors leads to chronic and sustained infections associated with worsening prognosis. In this study, we present the X-ray crystal structure of AmrZ in complex with DNA containing the repressor site, amrZ1. Binding of AmrZ to this site leads to auto-repression. AmrZ binds this DNA sequence as a dimer-of-dimers, and makes specific base contacts to two half sites, separated by a five base pair linker region. Analysis of the linker region shows a narrowing of the minor groove, causing significant distortions. AmrZ binding assays utilizing sequences containing variations in this linker region reveals that secondary structure of the DNA, conferred by the sequence of this region, is an important determinant in binding affinity. The results from these experiments allow for the creation of a model where both intrinsic structure of the DNA and specific nucleotide recognition are absolutely necessary for binding of the protein. We also examined AmrZ binding to the algD promoter, which results in activation of the alginate exopolysaccharide biosynthetic operon, and found the protein utilizes different interactions with this site. Finally, we tested the in vivo effects of this differential binding by switching the AmrZ binding site at algD, where it acts as an activator, for a repressor binding sequence and show that differences in binding alone do not affect transcriptional regulation.
Author Summary
The bacterium Pseudomonas aeruginosa causes a variety of human infections and is the leading cause of death in patients with cystic fibrosis. The main reason for the severity of these infections arises from the ability of P. aeruginosa to express virulence factors that protect it from the host immune system. Several of these processes are controlled by a transcription factor called AmrZ, a potential target for anti-microbial therapeutics. AmrZ is unusual in that it has the ability to both activate some genes, such as for alginate biofilm, and repress others, as with flagellum and itself. Here we determine the three dimensional structure of AmrZ bound to DNA containing a repressor sequence. Our structure shows the specific interactions the protein makes with the DNA for binding and repression. It also reveals that both the sequence and shape of the DNA are important for tight association. We next examined the binding of the protein to DNA containing an activator sequence and found that it has different interactions. However, by switching the AmrZ binding site at algD, where it acts as an activator, for a repressor binding sequence in P. aeruginosa, we show that differences in binding alone do not account for transcriptional regulation.
doi:10.1371/journal.ppat.1002648
PMCID: PMC3325190  PMID: 22511872
24.  A bacterial antirepressor with SH3 domain topology mimics operator DNA in sequestering the repressor DNA recognition helix 
Nucleic Acids Research  2010;38(15):5226-5241.
Direct targeting of critical DNA-binding elements of a repressor by its cognate antirepressor is an effective means to sequester the repressor and remove a transcription initiation block. Structural descriptions for this, though often proposed for bacterial and phage repressor–antirepressor systems, are unavailable. Here, we describe the structural and functional basis of how the Myxococcus xanthus CarS antirepressor recognizes and neutralizes its cognate repressors to turn on a photo-inducible promoter. CarA and CarH repress the carB operon in the dark. CarS, produced in the light, physically interacts with the MerR-type winged-helix DNA-binding domain of these repressors leading to activation of carB. The NMR structure of CarS1, a functional CarS variant, reveals a five-stranded, antiparallel β-sheet fold resembling SH3 domains, protein–protein interaction modules prevalent in eukaryotes but rare in prokaryotes. NMR studies and analysis of site-directed mutants in vivo and in vitro unveil a solvent-exposed hydrophobic pocket lined by acidic residues in CarS, where the CarA DNA recognition helix docks with high affinity in an atypical ligand-recognition mode for SH3 domains. Our findings uncover an unprecedented use of the SH3 domain-like fold for protein–protein recognition whereby an antirepressor mimics operator DNA in sequestering the repressor DNA recognition helix to activate transcription.
doi:10.1093/nar/gkq277
PMCID: PMC2926617  PMID: 20410074
25.  Temperature-sensitive mutations in the bacteriophage Mu c repressor locate a 63-amino-acid DNA-binding domain. 
Journal of Bacteriology  1991;173(20):6568-6577.
Phage Mu's c gene product is a cooperative regulatory protein that binds to a large, complex, tripartite 184-bp operator. To probe the mechanism of repressor action, we isolated and characterized 13 phage mutants that cause Mu to undergo lytic development when cells are shifted from 30 to 42 degrees C. This collection contained only four mutations in the repressor gene, and all were clustered near the N terminus. The cts62 substitution of R47----Q caused weakened specific DNA recognition and altered cooperativity in vitro. A functional repressor with only 63 amino acids of Mu repressor fused to a C-terminal fragment of beta-galactosidase was constructed. This chimeric protein was an efficient repressor, as it bound specifically to Mu operator DNA in vitro and its expression conferred Mu immunity in vivo. A DNA looping model is proposed to explain regulation of the tripartite operator site and the highly cooperative nature of repressor binding.
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PMCID: PMC208994  PMID: 1833382

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