Factor For Inversion Stimulation Research Articles

Regulation of adhE (encoding ethanol oxidoreductase) by the Fis protein in Escherichia coli.

The adhE gene of Escherichia coli encodes a multifunctional ethanol oxidoreductase whose expression is 10-fold higher under anaerobic than aerobic conditions. Transcription of the gene is under the negative control of the Cra (catabolite repressor-activator) protein, whereas translation of the adhE mRNA requires processing by RNase III. In this report, we show that the expression of adhE also depends on the Fis (factor for inversion stimulation) protein. A strain bearing a fis::kan null allele failed to grow anaerobically on glucose solely because of inadequate adhE transcription. However, fis expression itself is not under redox control. Sequence inspection of the adhE promoter revealed three potential Fis binding sites. Electrophoretic mobility shift analysis, using purified Fis protein and adhE promoter DNA, showed three different complexes.

Twelve Species of the Nucleoid-associated Protein from Escherichia coli: SEQUENCE RECOGNITION SPECIFICITY AND DNA BINDING AFFINITY

The genome of Escherichia coli is composed of a single molecule of circular DNA with the length of about 47,000 kilobase pairs, which is associated with about 10 major DNA-binding proteins, altogether forming the nucleoid. We expressed and purified 12 species of the DNA-binding protein, i.e. CbpA (curved DNA-binding protein A), CbpB or Rob (curved DNA-binding protein B or right arm of the replication origin binding protein), DnaA (DNA-binding protein A), Dps (DNA-binding protein from starved cells), Fis (factor for inversion stimulation), Hfq (host factor for phage Q(beta)), H-NS (histone-like nucleoid structuring protein), HU (heat-unstable nucleoid protein), IciA (inhibitor of chromosome initiation A), IHF (integration host factor), Lrp (leucine-responsive regulatory protein), and StpA (suppressor of td(-) phenotype A). The sequence specificity of DNA binding was determined for all the purified nucleoid proteins using gel-mobility shift assays. Five proteins (CbpB, DnaA, Fis, IHF, and Lrp) were found to bind to specific DNA sequences, while the remaining seven proteins (CbpA, Dps, Hfq, H-NS, HU, IciA, and StpA) showed apparently sequence-nonspecific DNA binding activities. Four proteins, CbpA, Hfq, H-NS, and IciA, showed the binding preference for the curved DNA. From the apparent dissociation constant (K(d)) determined using the sequence-specific or nonspecific DNA probes, the order of DNA binding affinity were determined to be: HU > IHF > Lrp > CbpB(Rob) > Fis > H-NS > StpA > CbpA > IciA > Hfq/Dps, ranging from 25 nM (HU binding to the non-curved DNA) to 250 nM (Hfq binding to the non-curved DNA), under the assay conditions employed.

Modeling Helix-Turn-Helix Protein-Induced DNA Bending with Knowledge-Based Distance Restraints

A crucial element of many gene functions is protein-induced DNA bending. Computer-generated models of such bending have generally been derived by using a presumed bending angle for DNA. Here we describe a knowledge-based docking strategy for modeling the structure of bent DNA recognized by a major groove-inserting α-helix of proteins with a helix-turn-helix (HTH) motif. The method encompasses a series of molecular mechanics and dynamics simulations and incorporates two experimentally derived distance restraints: one between the recognition helix and DNA, the other between respective sites of protein and DNA involved in chemical modification-enabled nuclease scissions. During simulation, a DNA initially placed at a distance was “steered” by these restraints to dock with the binding protein and bends. Three prototype systems of dimerized HTH DNA binding were examined: the catabolite gene activator protein (CAP), the phage 434 repressor (Rep), and the factor for inversion stimulation (Fis). For CAP-DNA and Rep-DNA, the root mean square differences between model and x-ray structures in nonhydrogen atoms of the DNA core domain were 2.5 Å and 1.6 Å, respectively. An experimental structure of Fis-DNA is not yet available, but the predicted asymmetrical bending and the bending angle agree with results from a recent biochemical analysis.

The FIS protein fails to block the binding of DnaA protein to oriC, the Escherichia coli chromosomal origin.

The Escherichia coli chromosomal origin contains several bindings sites for factor for inversion stimulation (FIS), a protein originally identified to be required for DNA inversion by the Hin and Gin recombinases. The primary FIS binding site is close to two central DnaA boxes that are bound by DnaA protein to initiate chromosomal replication. Because of the close proximity of this FIS site to the two DnaA boxes, we performed in situ footprinting with 1, 10-phenanthroline-copper of complexes formed with FIS and DnaA protein that were separated by native gel electrophoresis. These studies show that the binding of FIS to the primary FIS site did not block the binding of DnaA protein to DnaA boxes R2 and R3. Also, FIS appeared to be bound more stably to oriC than DnaA protein, as deduced by its reduced rate of dissociation from a restriction fragment containing oriC . Under conditions in which FIS was stably bound to the primary FIS site, it did not inhibit oriC plasmid replication in reconstituted replication systems. Inhibition, observed only at high levels of FIS, was due to absorption by FIS binding of the negative superhelicity of the oriC plasmid that is essential for the initiation process.

Defining the structural and functional roles of the carboxyl region of the bacteriophage lambda excisionase (Xis) protein

The bacteriophage λ excisionase (Xis) protein is required for excisive site-specific recombination. Xis is composed of 72 amino acids and binds cooperatively to two DNA sites (X1 and X2) that are arranged as direct repeats. Alternatively, Xis binds cooperatively with the host-encoded factor for inversion stimulation (FIS) protein at the X1 and F sites, respectively. Here we analyzed the effects of missense substitutions from codon 57 to the carboxyl end of the protein and nonsense mutations that truncate the protein at various positions from residues 60 to 69. We find that all of the mutant proteins promote excision to some extent and interact cooperatively with FIS. Some mutants have no detectible phenotype while others are altered in their abilities to promote excision or to interact cooperatively with integrase (Int). Computer modeling predicts that amino acids from residues 59 to 65 are in an α-helix conformation. Mutants with substitutions on one side of the helix at residues 57, 60, 63 and 64 as well as truncated mutants containing 60, 61 or 63 amino acids, fail to interact cooperatively with Int suggesting that this region of the protein forms the interface with Int. Mutants with substitutions at other positions in the putative helix have no detectible phenotype. Residues 66 to 68 may form a reverse turn and the last four amino acids (69 to 72) may not be crucial for the structure or function of the protein.

Influence of FIS on the transcription from closely spaced and non-overlapping divergent promoters for an aminoacyl-tRNA synthetase gene (gltX) and a tRNA operon (valU) in Escherichia coli.

The gltX gene, encoding the glutamyl-tRNA synthetase (GluRS), and the valU operon, whose transcripts contain three tRNAVal/UAC and one tRNALys/UUU, are adjacent and divergently transcribed. It is the only known case of adjacent genes encoding an aminoacyl-tRNA synthetase and a tRNA precursor in Escherichia coli. The gltX promoters (P1, P2 and P3) direct the synthesis of transcripts non-overlapping with and divergent from the one initiated at the valU promoter. We report that their promoter region (250 bp) contains three binding sites for the factor for inversion stimulation (FIS), centred at positions -71, -91 and -112 from the valU transcription initiation site, and that the destruction of any of these sites does not prevent the binding of FIS to the others. As FIS is one of the major positive regulators of stable RNA operons, we have studied its role on gltX and valU transcription. FIS stimulates valU transcription in vitro and about twofold in vivo during steady-state exponential growth. In contrast, gltX transcription is repressed by the presence of FIS in vitro and about twofold in vivo during growth acceleration when a decrease in GluRS concentration was observed. Under all conditions tested, most of the gltX transcripts start at the P3 promoter. Nested deletions of this regulatory region reveal that the FIS-dependent repression of the gltX-P3 promoter is abolished after the removal of the valU promoter, and is not altered by the additional removal of the FIS binding sites; moreover, in vivo transcription from gltX-P1 and/or gltX-P2 present on some of these regulatory region variants is modulated by the nature of the upstream region by FIS and is sometimes stronger than that from gltX-P3. These results show that the strength and the site of gltX transcription initiation are influenced by the upstream region up to and including the valU promoter; furthermore, they indicate that although these adjacent genes are involved in the first step of protein biosynthesis and share cis and trans regulatory elements, their transcription is non-co-ordinate.

Information analysis of Fis binding sites.

Originally discovered in the bacteriophage Mu DNA inversion system gin, Fis (Factor for Inversion Stimulation) regulates many genetic systems. To determine the base frequency conservation required for Fis to locate its binding sites, we collected a set of 60 experimentally defined wild-type Fis DNA binding sequences. The sequence logo for Fis binding sites showed the significance and likely kinds of base contacts, and these are consistent with available experimental data. Scanning with an information theory based weight matrix within fis, nrd, tgt/sec and gin revealed Fis sites not previously identified, but for which there are published footprinting and biochemical data. DNA mobility shift experiments showed that a site predicted to be 11 bases from the proximal Salmonella typhimurium hin site and a site predicted to be 7 bases from the proximal P1 cin site are bound by Fis in vitro. Two predicted sites separated by 11 bp found within the nrd promoter region, and one in the tgt/sec promoter, were also confirmed by gel shift analysis. A sequence in aldB previously reported to be a Fis site, for which information theory predicts no site, did not shift. These results demonstrate that information analysis is useful for predicting Fis DNA binding.

Role of architectural elements in combinatorial regulation of initiation of DNA replication in Escherichia coli.

Bending of DNA is a prerequisite of site-specific recombination and gene expression in many regulatory systems involving the assembly of specific nucleoprotein complexes. We have investigated how the uniquely clustered Dam methylase sites, GATCs, in the origin of Escherichia coli replication (oriC) and their methylation status modulate the geometry of oriC and its interaction with architectural proteins, such as integration host factor (IHF), factor for inversion stimulation (Fis) and DnaA initiator protein. We note that 3 of the 11 GATC sites at oriC are strategically positioned within the IHF protected region. Methylation of the GATCs enhances IHF binding and alters the IHF-induced bend at oriC. GATC motifs also contribute to intrinsic DNA curvature at oriC and the degree of bending is modulated by methylation. The IHF-induced bend at oriC is further modified by Fis protein and IHF affinity for its binding site may be impaired by protein(s) binding to GATCs within the IHF site. Thus, GATC sites at oriC affect the DNA conformation and GATCs, in conjunction with the protein-induced bends, are critical cis-acting elements in specifying proper juxtapositioning of initiation factors in the early steps of DNA replication.

FIS activates sequential steps during transcription initiation at a stable RNA promoter.

FIS (factor for inversion stimulation) is a small dimeric DNA-bending protein which both stimulates DNA inversion and activates transcription at stable RNA promoters in Escherichia coli. Both these processes involve the initial formation of a complex nucleoprotein assembly followed by local DNA untwisting at a specific site. We have demonstrated previously that at the tyrT promoter three FIS dimers are required to form a nucleoprotein complex with RNA polymerase. We now show that this complex is structurally dynamic and that FIS, uniquely for a prokaryotic transcriptional activator, facilitates sequential steps in the initiation process, enabling efficient polymerase recruitment, untwisting of DNA at the transcription startpoint and finally the escape of polymerase from the promoter. Activation of all these steps requires that the three FIS dimers bind in helical register. We suggest that FIS acts by stabilizing a DNA microloop whose topology is coupled to the local topological transitions generated during the initiation of transcription.

FIS is a regulator of metabolism in Escherichia coli.

The Escherichia coli DNA-binding protein FIS (factor for inversion stimulation) stimulates site-specific recombination reactions catalysed by DNA invertases and is an activator of stable RNA synthesis. To address the question of whether FIS is involved in other cellular processes we have identified and sequenced proteins whose expression pattern is affected by FIS. This has led to the identification of several E. coli genes whose expression in vivo is either enhanced or repressed by FIS. All of these genes encode enzymes or transport proteins involved in the catabolism of sugars or nucleic acids, and their expression is also dependent on the cAMP-CRP complex. In most cases studied the regulation by FIS is indirect and occurs through effects on the synthesis of the respective repressor proteins. We conclude that FIS is a transcriptional modulator involved in the regulation of metabolism in E. coli.

Structure model of a complex between the factor for inversion stimulation (FIS) and DNA: modeling protein-DNA complexes with dyad symmetry and known protein structures.

A method is presented to predict overall conformations of protein-DNA complexes on the basis of the known three-dimensional structures of the proteins. The method is restricted to proteins with a common twofold symmetry axis, which show only minor conformational changes upon binding to DNA. The method uses a numerical finite difference solution of the linearized Poisson-Boltzmann equation and subsequent energy minimization cycles. Structural parameters-the rotation angle of the DNA relative to the protein around the common symmetry axis, the protein-DNA distance, and intermolecular hydrogen-bonding contacts-are presented for two test cases, DNA bound to CAP (catabolite gene activator protein) and to the Cro-repressor of bacteriophage 434. The DNA curvature in the starting model of the docking procedure was chosen as a smoothed approximation of the conformation found in the X-ray structures of these complexes. The method is further used to predict the unknown structure of the complex between the factor for inversion stimulation (FIS) and DNA, which is bent upon binding to FIS. In contrast to the test cases, the unknown curvature of the starting model is derived from a calibration of electrostatic precalculations for different proteins according to crystallographically observed DNA bending. The results of the modeling are in good accordance with the experimentally observed overall structure of protein-DNA complexes for the two test cases; for FIS, they correspond to several of the experimentally proposed protein-DNA contacts.

Structure model of a complex between the factor for inversion stimulation (FIS) and DNA: Modeling protein-DNA complexes with dyad symmetry and known protein structures

A method is presented to predict overall conformations of protein-DNA complexes on the basis of the known three-dimensional structures of the proteins. The method is restricted to proteins with a common twofold symmetry axis, which show only minor conformational changes upon binding to DNA. The method uses a numerical finite difference solution of the linearized Poisson-Boltzmann equation and subsequent energy minimization cycles. Structural parameters—the rotation angle of the DNA relative to the protein around the common symmetry axis, the protein-DNA distance, and intermolecular hydrogen-bonding contacts—are presented for two test cases, DNA bound to CAP (catabolite gene activator protein) and to the Cro-repressor of bacteriophage 434. The DNA curvature in the starting model of the docking procedure was chosen as a smoothed approximation of the conformation found in the X-ray structures of these complexes. The method is further used to predict the unknown structure of the complex between the factor for inversion stimulation (FIS) and DNA, which is bent upon binding to FIS. In contrast to the test cases, the unknown curvature of the starting model is derived from a calibration of electrostatic precalculations for different proteins according to crystallographically observed DNA bending. The results of the modeling are in good accordance with the experimentally observed overall structure of protein-DNA complexes for the two test cases; for FIS, they correspond to several of the experimentally proposed protein-DNA contacts. © 1996 Wiley-Liss, Inc.

Structure model of a complex between the factor for inversion stimulation (FIS) and DNA: Modeling protein‐DNA complexes with dyad symmetry and known protein structures

Structure model of a complex between the factor for inversion stimulation (FIS) and DNA: Modeling protein‐DNA complexes with dyad symmetry and known protein structures

The nd7‐binding protein (Nbp) regulates the ndh gene of Escherichia coti in response to growth phase and is identical to Fis

The ndh gene that encodes the non-proton-translocating NADH dehydrogenase II of Escherichia coli is anaerobically repressed by FNR. However, in the absence of FNR, ndh expression is enhanced by anaerobic growth in media containing amino acids. Two potential regulatory proteins that may be associated with this activation have previously been detected, Arr (amino acid response regulator) and Nbp (ndh-binding protein). Studies with the heat-stable Nbp have now shown that it is present in E. coli grown both aerobically and anaerobically in rich and minimal media, indicating that it is not specifically associated with the anaerobic enhancement of ndh expression. The Nbp activity of aerobic cultures was maximal during exponential growth phase (when ndh promoter activity is minimal) but fell rapidly as cultures entered stationary phase and ndh expression increased. Protein purification and mutant studies have further shown that Nbp is identical to the Fis protein (factor for inversion stimulation). Three major and two minor Nbp (Fis)-binding sites have been identified in the ndh promoter by gel retardation and DNase I footprinting. The major sites are centred at -123, -72 and +51, in decreasing order of binding affinity. At low concentrations, Nbp (Fis) increased transcription from the ndh promoter by up to 25%, whereas at higher concentrations it prevented RNA polymerase (RNAP) binding and open complex formation. Consequently, Nbp (Fis) can both activate and repress transcription from the ndh promoter. The results suggest that Nbp (Fis) serves to ensure that the energetically efficient proton-translocating NADH dehydrogenase I is used in preference to the non-proton translocating NADH dehydrogenase II during periods of rapid growth, by repressing expression of the ndh gene.

Antagonistic involvement of FIS and H-NS proteins in the transcriptional control of hns expression.

Gel shift and DNase I footprinting experiments showed that Escherichia coli FIS (factor for inversion stimulation) protein binds to at least seven sites in the promoter region of hns. These sites extend from -282 to +25 with two sites, closely flanking the DNA bend located at -150 from the transcriptional startpoint, partly overlapping the H-NS binding sites involved in the transcriptional autorepression of hns. The interplay between FIS, H-NS and the hns promoter region were studied by examining the effects of FIS and H-NS on in vitro transcription of hns-cat fusions, as well as looking at the effect of FIS on preformed complexes containing H-NS and a DNA fragment derived from the hns promoter region. Taken together, our data suggest that in the cell, FIS and H-NS interact with the promoter region of hns and influence their respective interactions (possibly competing for the same binding site), eliciting antagonistic effects so that an interplay between these proteins might contribute to the transcriptional control of hns.

Cell cycle-specific changes in nucleoprotein complexes at a chromosomal replication origin.

Initiation of DNA synthesis is triggered by the binding of proteins to replication origins. However, little is known about the order in which specific proteins associate with origin sites during the cell cycle. We show that in cycling cells there are at least two different nucleoprotein complexes at oriC. A factor for inversion stimulation (FIS)-bound nucleoprotein complex, present throughout the majority of the cell cycle, switches to an integration host factor (IHF)-bound form as cells initiate DNA replication. Coincident with binding of IHF, initiator DnaA binds to its previously unoccupied R3 site. In stationary phase, a third nucleoprotein complex forms. FIS is absent and inactive oriC forms a nucleoprotein structure containing IHF that is not observed in cycling cells. We propose that interplay between FIS and IHF aids assembly of initiation nucleoprotein complexes during the cell cycle and blocks initiation at inappropriate times. This exchange of components at replication origins is reminiscent of switching between pre- and post-replicative chromatin states at yeast ARS1.

The Major Dimerization Determinants of the Nitrogen Regulatory Protein NTRC from Enteric Bacteria Lie in Its Carboxy-terminal Domain

The NTRC protein (nitrogen regulatory protein C) of enteric bacteria is an enhancer-binding protein that activates transcription by the σ54-holoenzyme form of RNA polymerase. NTRC is a homodimeric protein that binds to a dyad-symmetrical site in DNA. To activate transcription NTRC must be phosphorylated and must form an appropriate oligomeric species at an enhancer.In order to study subunit exchange between NTRC dimers, we constructed a fusion of the maltose-binding protein (MBP) to the amino-terminal end of NTRC (MBP-NTRC) and visualized the formation of heterodimers between MBP-NTRC and wild-type NTRC by a gel-mobility shift assay for DNA-binding. When MBP-NTRC is mixed with wild-type NTRC at 37°C, subunit exchange occurs rapidly. The apparent half-life for dissociation of homodimers of NTRC is two to three minutes at 37°C and is not changed by phosphorylation.The isolated carboxy-terminal domain of NTRC (91 amino acid residues) forms heterodimers with both wild-type NTRC and MBP-NTRC, indicating that the C-terminal domain is sufficient for dimerization. The apparent rate of dissociation of homodimers of the C-terminal domain is essentially the same as that of full-length NTRC, indicating that the major dimerization determinants of the protein lie in its C-terminal domain. Congruent with this, a truncated form of NTRC from which the last 58 amino acid residues were removed is a monomer in solution. Moreover, truncated forms of NTRC from which the last 16 or 26 amino acid residues were removed are predominantly monomeric in solution, as is a mutant form with the amino acid substitution A410E in its C-terminal domain Monomerization of the above mutant forms of NTRC can be rationalized on the basis of homology between the C-terminal region of NTRC and a 50 amino acid residue region of the factor for inversion stimulation (FIS) protein.

Factor for inversion stimulation-dependent growth rate regulation of individual tRNA species in Escherichia coli

We have studied the involvement of the factor for inversion stimulation (FIS) in the growth rate-dependent expression of the arginine, leucine, and methionine acceptor tRNA species. The concentration of individual tRNA species relative to 16 S rRNA was determined by blot hybridization using RNA preparations from bacteria with the fis gene deleted and from isogenic wild type bacteria. The RNA preparations were obtained from bacteria growing under steady state conditions in different media. The levels of tRNA(1Leu), tRNA(2Arg), tRNA(4Arg), and tRNA(5Arg decreased in the fis bacteria, relative to the wild type. The difference in levels increased with increasing growth rate. Surprisingly, tRNA(3Leu), tRNA(rMet), and tRNA(eMet) showed the opposite response, with an increase of the tRNA/16 S ratio in the fis bacteria. The tRNA(2Leu, tRNA(4Leu), tRNA(5Leu), and tRNA(3 Arg) had unaffected tRNA/16 S ratios in fis cells. We conclude that FIS, directly or indirectly, is involved in growth rate regulation of some tRNA species and that it affects the composition of the cellular tRNA pool.

Structure of the Escherichia coli Fis-DNA complex probed by protein conjugated with 1,10-phenanthroline copper(I) complex.

The Escherichia coli Fis (factor for inversion stimulation) protein functions in many diverse biological systems including recombination, transcription, and DNA replication. Although Fis is a site-specific DNA-binding protein, it lacks a well-defined consensus recognition sequence. The electrophoretic mobility of Fis-DNA complexes, along with considerations of the Fis crystal structure, indicates that significant deformation of DNA occurs upon Fis binding. To investigate the structure of Fis-DNA complexes, the chemical nuclease 1,10-phenanthroline-copper complex (OP-Cu) has been linked to four specific sites within the Fis DNA-binding domain. Two of these Fis-OP derivatives were active in cleaving DNA. The scission patterns obtained on four different Fis binding sites indicate that Fis positions itself on these highly divergent DNA sequences in a very similar fashion. The patterns of cleavage of a derivative at Asn-98 generally support a model of a Fis-DNA complex that contains specific bends within the core-recognition sequence. Data from a second Fis-OP derivative at Asn-73 provides evidence for greater wrapping of flanking DNA around the sides of the Fis protein than was previously postulated. The cleavage efficiency of flanking segments varies, suggesting that the extent of DNA wrapping is sequence dependent. Specific amino acids on Fis are implicated in promoting this DNA wrapping.

Involvement of Escherichia coli FIS protein in maintenance of bacteriophage mu lysogeny by the repressor: control of early transcription and inhibition of transposition.

The Escherichia coli FIS (factor for inversion stimulation) protein has been implicated in assisting bacteriophage Mu repressor, c, in maintaining the lysogenic state under certain conditions. In a fis strain, a temperature-inducible Mucts62 prophage is induced at lower temperatures than in a wild-type host (M. Bétermier, V. Lefrère, C. Koch, R. Alazard, and M. Chandler, Mol. Microbiol. 3:459-468, 1989). Increasing the prophage copy number rendered Mucts62 less sensitive to this effect of the fis mutation, which thus seems to depend critically on the level of repressor activity. The present study also provides evidence that FIS affects the control of Mu gene expression and transposition. As judged by the use of lac transcriptional fusions, repression of early transcription was reduced three- to fourfold in a fis background, and this could be compensated by an increase in cts62 gene copy number. c was also shown to inhibit Mu transposition two- to fourfold less strongly in a fis host. These modulatory effects, however, could not be correlated to sequence-specific binding of FIS to the Mu genome, in particular to the strong site previously identified on the left end. We therefore speculate that a more general function of FIS is responsible for the observed modulation of Mu lysogeny.