Repression Loop Research Articles

Proteolytic Excision of a Repressive Loop Domain in Tartrate-resistant Acid Phosphatase by Cathepsin K in Osteoclasts

Tartrate-resistant acid phosphatase (TRAP) is a metallophosphoesterase participating in osteoclast-mediated bone turnover. Activation of TRAP is associated with the redox state of the di-iron metal center as well as with limited proteolytic cleavage in an exposed loop domain. The cysteine proteinases cathepsin B, L, K, and S as well as the matrix metalloproteinase-2, -9, -13, and -14 are expressed by osteoclasts and/or other bone cells and have been implicated in the turnover of bone and cartilage. To identify proteases that could act as activators of TRAP in bone, we report here that cathepsins K and L, in contrast to the matrix metalloproteinases, efficiently cleaved and activated recombinant TRAP in vitro. Activation of TRAP by cathepsin K/L was because of increases in catalytic activity, substrate affinity, and sensitivity to reductants. Processing by cathepsin K occurred sequentially by an initial excision of the loop peptide Gly(143)-Gly(160) followed by the removal of a Val(161)-Ala(162) dipeptide at the N terminus of the C-terminal 16-kDa TRAP subunit. Cathepsin L initially released a shorter Gln(151)-Gly(160) peptide and completed processing at Ser(145) or Gly(143) at the C terminus of the N-terminal 23-kDa TRAP subunit and at Arg(163) at the N terminus of the C-terminal 16-kDa TRAP subunit. Mutation of Ser(145) to Ala partly mimicked the effect of proteolysis on catalytic activity, identifying Ser(145) as well as Asp(146) (Funhoff, E. G., Ljusberg, J., Wang, Y., Andersson, G., and Averill, B. A. (2001) Biochemistry 40, 11614-11622) as repressive amino acids of the loop region to maintain the TRAP enzyme in a catalytically latent state. The C-terminal sequence of TRAP isolated from rat bone was consistent with cathepsin K-mediated processing in vivo. Moreover, cathepsin K, but not cathepsin L, co-localized with TRAP in osteoclast-resorptive compartments, supporting a role for cathepsin K in the extracellular processing of monomeric TRAP in the resorption lacuna.

Bacterial Repression Loops Require Enhanced DNA Flexibility

The Escherichia coli lac operon provides a classic paradigm for understanding regulation of gene transcription. It is now appreciated that lac promoter repression involves cooperative binding of the bidentate lac repressor tetramer to pairs of lac operators via DNA looping. We have adapted components of this system to create an artificial assay of DNA flexibility in E.coli. This approach allows for systematic study of endogenous and exogenous proteins as architectural factors that enhance apparent DNA flexibility in vivo. We show that inducer binding does not completely remove repression loops but it does alter their geometries. Deletion of the E.coli HU protein drastically destabilizes small repression loops, an effect that can be partially overcome by expression of a heterologous mammalian HMG protein. These results emphasize that the inherent torsional inflexibility of DNA restrains looping and must be modulated in vivo.

Crystal Structures of Recombinant Human Purple Acid Phosphatase With and Without an Inhibitory Conformation of the Repression Loop

The crystal structure of human purple acid phosphatase recombinantly expressed in Escherichia coli (rHPAP(Ec)) and Pichia pastoris (rHPAP(Pp)) has been determined in two different crystal forms, both at 2.2A resolution. In both cases, the enzyme crystallized in its oxidized (inactive) state, in which both Fe atoms in the dinuclear active site are Fe(III). The main difference between the two structures is the conformation of the enzyme "repression loop". Proteolytic cleavage of this loop in vivo or in vitro results in significant activation of the mammalian PAPs. In the crystals obtained from rHPAP(Ec), the carboxylate side-chain of Asp145 of this loop acts as a bidentate ligand that bridges the two metal atoms, in a manner analogous to a possible binding mode for a phosphate ester substrate in the enzyme-substrate complex. The carboxylate side-chain of Asp145 and the neighboring Phe146 side-chain thus block the active site, thereby inactivating the enzyme. In the crystal structure of rHPAP(Pp), the enzyme "repression loop" has an open conformation similar to that observed in other mammalian PAP structures. The present structures demonstrate that the repression loop exhibits significant conformational flexibility, and the observed alternate binding mode suggests a possible inhibitory role for this loop.

N-glycosylation influences the latency and catalytic properties of mammalian purple acid phosphatase

Purple acid phosphatase (PAP), also known as tartrate-resistant acid phosphatase or uteroferrin, contains two potential consensus N-glycosylation sites at Asn 97 and Asn 128. In this study, endogenous rat bone PAP was found to possess similar N-glycan structures as rat recombinant PAP heterologously expressed in baculovirus-infected Sf9 insect cells. PAP from Sf9 cells was shown to contain two N-linked oligosaccharides, whereas PAP expressed by mammalian CHO-K1 cells was less extensively glycosylated. The extent of N-glycosylation affected the catalytic properties of the enzyme, as N97Q and N128Q mutants, containing a single oligosaccharide chain, exhibited a lower substrate affinity and catalytic activity compared to those of the fully glycosylated PAP in the native, monomeric state. The differences in substrate affinity and catalytic activity were abolished and partially restored, respectively, by proteolytic cleavage in the loop domain, indicating that the extent of N-glycosylation influences the interaction of the repressive loop domain with catalytically important residues.

Repression of the Escherichia coli melR promoter by MelR: evidence for a repression loop

Repression of the <i>Escherichia coli melR</i> promoter by MelR: evidence for a repression loop

Repression of the Escherichia coli melR promoter by MelR: evidence that efficient repression requires the formation of a repression loop.

The Escherichia coli MelR protein is a transcription activator that, in the presence of melibiose, activates expression of the melAB operon by binding to four sites located just upstream of the melAB promoter. MelR is encoded by the melR gene, which is expressed from a divergent transcript that starts 237 bp upstream of the melAB promoter transcript start point. In a recent study, we have identified a fifth DNA site for MelR that overlaps the melR promoter transcript start and -10 region. Here we show that MelR binding to this site can downregulate expression from the melR promoter; thus, MelR autoregulates its own expression. Optimal repression of the melR promoter is observed in the absence of melibiose and requires one of the four other DNA sites for MelR at the melAB promoter. The two MelR binding sites required for this optimal repression are separated by 177 bp. We suggest that, in the absence of melibiose, MelR forms a loop between these two sites. We argue that, in the presence of melibiose, this loop is broken as the melAB promoter is activated. However, in the presence of melibiose, the melR promoter can still be partially repressed by MelR binding to the site that overlaps the transcript start and -10 region. Parallels with the Escherichia coli araC-araBAD regulatory region are discussed.

DNA bending and expression of the divergent nagE-B operons.

Repression of the divergent nagE - B operons requires NagC binding to two operators which overlap the nagE and nagB promoters, resulting in formation of a DNA loop. Binding of the cAMP/CAP activator to its site, adjacent to the nagE operator, stabilizes the DNA loop in vitro. The DNA of the nagE-B intergenic region is intrinsically bent, with the bend centred on the CAP site. We show that displacement of the CAP site by 6 bp results in complete derepression of the two operons. This derepression is observed even in the absence of cAMP/CAP binding and despite the fact that the two NagC operators are still in phase, demonstrating that the inherently bent structure of the DNA loop is important for repression. Since no interaction between NagC and CAP has been detected, we propose that the role of CAP in the repression loop is architectural, stabilizing the intrinsic bend. The cAMP/CAP complex is necessary for activation of the nagE-B promoters. In this case protein-protein contacts between CAP and RNA polymerase are necessary for full activation, but at least a part of the activation is likely due to an effect of CAP binding altering DNA structure.

Action at a distance for negative control of transcription of the glpD gene encoding sn-glycerol 3-phosphate dehydrogenase of Escherichia coli K-12.

Aerobic sn-glycerol 3-phosphate dehydrogenase is a cytoplasmic membrane-associated respiratory enzyme encoded by the glpD gene of Escherichia coli. The glpD operon is tightly controlled by cooperative binding of the glp repressor to tandem operators (O(D)1 and O(D)2) that cover the -10 promoter element and 30 bp downstream of the transcription start site. In this work, two additional operators were identified within the glpD structural gene at positions 568 to 587 (0(D)3) and 609 to 628 (0(D)4). The two internal operators bound the glp repressor in the presence or absence of the tandem operators (O(D)1 and O(D)2) in vitro, as shown by DNase I footprinting. To assess a potential regulatory role for the two internal operators in vivo, a glpD-lacZ transcriptional fusion containing all four operators was constructed. The response of this fusion to the glp repressor was compared with those of fusion constructs in which O(D)3 and O(D)4 were inactivated by either deletion or site-directed mutagenesis. It was found that the repression conferred by binding of the glp repressor to O(D)1 and O(D)2 was increased five- to sevenfold upon introduction of the internal operators. A regulatory role for HU was suggested when it was found that repressor-mediated control of glpD transcription was increased fourfold in strains containing HU compared with that of strains deficient in HU. The effect of HU was apparent only in the presence of all four glpD operators. The results suggest that glpD is controlled by formation of a repression loop between the tandem and internal operators. HU may assist repression by bending the DNA to facilitate loop formation.

Crystal structure of the lactose operon repressor and its complexes with DNA and inducer.

The lac operon of Escherichia coli is the paradigm for gene regulation. Its key component is the lac repressor, a product of the lacI gene. The three-dimensional structures of the intact lac repressor, the lac repressor bound to the gratuitous inducer isopropyl-beta-D-1-thiogalactoside (IPTG) and the lac repressor complexed with a 21-base pair symmetric operator DNA have been determined. These three structures show the conformation of the molecule in both the induced and repressed states and provide a framework for understanding a wealth of biochemical and genetic information. The DNA sequence of the lac operon has three lac repressor recognition sites in a stretch of 500 base pairs. The crystallographic structure of the complex with DNA suggests that the tetrameric repressor functions synergistically with catabolite gene activator protein (CAP) and participates in the quaternary formation of repression loops in which one tetrameric repressor interacts simultaneously with two sites on the genomic DNA.

‘Muprints’ of the lac operon demonstrate physiological control over the randomness of in vivo transposition

A method called Muprinting has been developed that uses PCR to generate a detailed picture of the bacteriophage Mu transposition sites in chosen domains of the bacterial chromosome. Muprinting experiments in Escherichia coli show that the frequency of phage integration changes dramatically near two repressor binding sites in the lac operon. When the lac operon was repressed, hotspots for Mu transposition were found near the O1 and O2 operators that are proposed to make a repression loop. When cells were grown in lactose, Mu transposition near these operators was greatly diminished. Striking changes in transposition frequencies were limited to the control region and were not found in a region of the lacZ gene lying beyond the O2 operator. Muprints of the bgl operon showed a different pattern; hotspots for Mu transposition detected in sequences upstream of the bglC promoter when the operon was silenced changed when the operon became activated by mutation. By targeting transposition to the regulatory regions around non-expressed genes, Mu may demonstrate a self-restraint mechanism that allows the virus to move through its host genome without disrupting the functions that contribute to a healthy cell physiology.

Interaction at a distance between multiple operators controls the adjacent, divergently transcribed glpTQ-glpACB operons of Escherichia coli K-12.

The glp regulon of Escherichia coli encodes the proteins required for utilization of sn-glycerol 3-phosphate and its precursors. Transcription of the divergently transcribed glpTQ and glpACB operons is initiated at sites separated by 132 base pairs (bp) of DNA. These operons are controlled negatively by glp repressor and positively by the cAMP-cAMP receptor protein (CRP) complex. The locations of the binding sites for the glp repressor and for cAMP.CRP in the control regions of these operons were determined by DNase I footprinting. Binding of the glp repressor protected the region -32 to -51 (OT) in the glpTQ promoter, which was also the binding site for cAMP.CRP. Four repressor binding sites (-41 to -60 (OA1), -9 to -28 (OA2), +12 to -8 (OA3), and +52 to +33 (OA4)) and two cAMP.CRP binding sites (+11 to -11 and -30 to -51) were found in the glpACB promoter region. Comparison of the sequences of the repressor binding sites found in the glpTQ-glpACB control region with those operators previously described in the glpD operon allowed formulation of a consensus operator sequence which was the palindrome 5'-WATGTTCGWTAWC-GAACATW-3' (W is A or T). The role of each operator was assessed by measuring repression in constructs where individual operators were altered by site-directed mutagenesis. Alteration of OT did not significantly decrease repression of either operon. Each of the glpACB operators contributed to repression of both operons. These results suggest involvement of glpACB operator(s) in control of glpTQ expression perhaps via formation of a repression loop. Evidence supporting this hypothesis was obtained by measuring the degree of repression of the glpTQ promoter in constructs containing 6- or 10-bp insertions between the glpTQ and glpACB operators. A 6-bp insertion located within OA2 or between OT and OA1 eliminated repression of the glpTQ promoter, whereas significant repression was maintained in the case of a 10-bp insertion within OA2.

Purification and initial characterization of AhrC: the regulator of arginine metabolism genes in Bacillus subtilis.

The arginine-dependent repressor-activator from Bacillus subtilis, AhrC, has been overexpressed in Escherichia coli and purified to homogeneity. AhrC, expressed in E. coli, is able to repress a Bacillus promoter (argCp), which lies upstream of the argC gene. The purified protein is a hexamer with a subunit molecular mass of 16.7 kDa. Its ability to recognize DNA has been examined in vitro using argCp in both DNase I and hydroxyl radical protection assays. AhrC binds at two distinct sites within the argCp fragment. One site, argCo1, with the highest affinity for protein, is located within the 5' promoter sequences, whilst the other, argCo2, is within the coding region of argC. The data are consistent with the binding of a single hexamer of AhrC to argCo1 via four of its subunits, possibly allowing the remaining two subunits to bind at argCo2 in vivo forming a repression loop similar to those observed for the E. coli Lac repressor.

Cis-acting ompF mutations that result in OmpR-dependent constitutive expression.

OmpR and EnvZ differentially control the transcription of the major outer membrane porin genes, ompF and ompC, in Escherichia coli in response to the osmolarity of the medium. We have previously provided evidence that OmpR works both positively and negatively at the ompF promoter to give the characteristic switch from OmpF to OmpC production with increasing osmolarity. Here, we describe the isolation of cis-acting ompF mutations that affect negative regulation by OmpR by affecting the three-dimensional structure of the promoter region as measured by agarose gel mobility. These results further clarify the mechanism by which OmpR negatively regulates ompF expression, suggesting a model in which OmpR forms a repressive loop in the ompF promoter region.

AraC-DNA looping: Orientation and distance-dependent loop breaking by the cyclic AMP receptor protein

The arabinose operon promoter, p bad, is negatively regulated in the absence of arabinose by AraC protein, which forms a DNA loop by binding to two sites separated by 210 base-pairs, ara0 2 and araI 1 . p bad is also positively regulated by AraC—arabinose and the cyclic AMP receptor protein, CRP. We provide evidence that CRP breaks the ara0 2—araI 1 repression loop in vitro. The ability of CRP to break the loop in vitro and to activate p bad in vivo is dependent upon the orientation and distance of the CRP binding site relative to araI 1 . An insertion of one DNA helical turn, 11 base-pairs, between CRP and araI only partially inhibits CRP loop breaking and activation of p bad, while an insertion of less than one DNA helical turn, 4 base-pairs, not only abolishes CRP activation and loop breaking, but actually causes CRP to stabilize the loop and increases the araO 2 -mediated repression of p bad. Both integral and non-integral insertions of greater than one helical turn completely abolish CRP activation and loop breaking in vitro.

Dual mechanism of repression at a distance in the lac operon.

The mechanism by which the internal lacZ gene sequence O2 influences lac repression was investigated by using in vivo footprinting of operon mutants. Quantitative in vivo binding curves show that O2 strengthens by approximately 3-fold repressor binding to O1 that is located 400 base pairs upstream at the transcription start site. The internal O2 sequence also contributes to repression by a second mechanism: repressor bound internally blocks elongation of beta-galactosidase gene expression. This secondary mechanism of repression is facilitated by the remote O1 operator that strengthens binding to O2 12-fold. Thus, lac repression involves two mechanisms, both of which involve cooperation between remote operator elements. During mild repression only the initiation mechanism applies, but more severe repression favors formation of the presumptive O1-O2 repression loop that allows both mechanisms to act simultaneously.

Alternative DNA loops regulate the arabinose operon in Escherichia coli.

The araCBAD regulatory region of Escherichia coli contains two divergently oriented promoters and three sites to which AraC, the regulatory protein of the operon, can bind. This paper presents the results of in vivo dimethyl sulfate "footprinting" experiments to monitor occupancy of the three AraC sites and measurements of activity of the two promoters. These measurements were made both in the absence of the inducer arabinose and at various times after arabinose addition to growing cells containing the wild-type ara regulatory region or the regulatory region containing various deletions and point mutations. The data lead to the conclusion that two different DNA loops can form in the ara regulatory region. These loops are generated by AraC protein molecules binding to two different DNA sites and binding to each other. One of these loops predominates in the absence of arabinose and plays a major role in repressing activity of one of the promoters. Upon the addition of arabinose the amount of the first loop type, the repression loop, decreases and the amount of a second loop increases. Formation of this second loop precludes the counterproductive formation of the repression loop.

Probing co-operative DNA-binding in vivo: The lac O 1: O 3 interaction

The lac primary (O 1) and weak upstream pseudo (O 3) operators contained on a plasmid were footprinted in vivo in order to determine whether they act co-operatively in binding lac repressor in the cell. The occupancy at O 3 by lac repressor was substantially reduced upon deletion of the lac primary operator, demonstrating co-operativity at a distance. Plots of operator occupancy versus active repressor concentration were obtained for each operator by treating the cells with different amounts of the lac inducer isopropyl-β- d-thiogalactoside and probing lac repressor binding. This analysis can be used to obtain relative binding constants in vivo and demonstrates that O 3 binds repressor only 10·3-fold less tightly than O 1 in their co-operative interaction. The removal of DNA torsional tension in vivo by the use of coumermycin leads to the same loss of binding at O 3 as does deleting O 1. These in-vivo results are analogous to the in-vitro situation, where O 3 binds repressor strongly in a DNA repression loop only on supercoiled templates.

DNA supercoiling promotes formation of a bent repression loop in lac DNA

Titration experiments on supercoiled lac DNA show that one repressor tetramer can bind simultaneously to the primary lac operator and to the very weak lac pseudo-operator, located 93 base-pairs apart. The formation of this complex is accompanied by the appearance of an extreme hypersensitive site in a five base-pair sequence located approximately midway between the operators. This remote sequence is hypersensitive to attack by two different chemical probes, dimethyl sulfate and potassium permanganate, the latter of which is a new probe for distorted DNA. We interpret these results in terms of a complex in which lac repressor holds two remote operators together in a DNA loop. The formation of this bent DNA loop requires negative DNA supercoiling. In vivo, both lac operators bind repressor even though the presence of multiple operator copies has forced the two operators to compete for a limited amount of repressor. This suggests that the operator and pseudo-operator have similar affinities for repressor in vivo. Such similar affinities were observed in vitro only when DNA supercoiling forced formation of a repression loop.