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At least 55 records · Page 3Linked to original sources
PubMed1997

Stabilization of compact spermidine nucleoids from Escherichia coli under crowded conditions: implications for in vivo nucleoid structure.

Nucleoids from Escherichia coli were isolated in the presence of spermidine at low salt concentrations. The nucleoids denature at relatively low temperatures or salt concentrations, yielding broad slowly sedimenting zones and/or macroscopic aggregates upon sucrose gradient centrifugation. Denaturation is accompanied by a loss of a characteristically compact shape as visualized by light and electron microscopy. Addition of polyethylene glycol or dextran prevents these changes, extending the range of stability of the isolated nucleoids to temperatures and ionic conditions like those which commonly occur in vivo. The effects of the polymers are consistent with stabilization by macromolecular crowding. Enzymatic digestion of the nucleoid DNA primarily releases three small proteins (H-NS, FIS, and HU) and RNA polymerase, as well as residual lysozyme from the cell lysis procedure. If isolated nucleoids are extracted with elevated salt concentrations under crowded, stabilized conditions, two of the proteins (HU and lysozyme) are efficiently removed and the compact form of the nucleoids is retained. These extracted nucleoids maintain their compact form upon reisolation into the initial uncrowded low-salt medium, indicating that HU, the most common "histone-like" protein of E. coli, is not a necessary component for maintaining compaction in these preparations.

Bacterial Proteins↗
PubMed1992

The complex for replication initiation of Escherichia coli.

We probed the complex between oriC and DnaA protein using two types of mutants in oriC. Base changes in the DnaA binding sites, DnaA boxes, had little effect on origin function. Mutations which change the distance between DnaA boxes R3 and R4, on the other hand, inactivated oriC unless the mutation deleted or inserted one complete helical turn. Origins with other 10 base pair insertions in the interval between DnaA boxes R2 and R3 were functional, but not insertions in the R1-R2 interval. FIS protein binds to a bipartite site in oriC between DnaA boxes R2 and R3. A model for the oriC/DnaA complex based on these results suggests an array of DnaA monomers with a 34 A spacing upon which oriC is arranged.

Bacterial Proteins↗
PubMed2002

FIS modulates the kinetics of successive interactions of RNA polymerase with the core and upstream regions of the tyrT promoter.

We have applied laser UV photo-footprinting to characterise kinetically complexes involving the activator protein FIS, RNA polymerase and the tyrT promoter of Escherichia coli. FIS photo-footprints strongly to three binding sites upstream of the core promoter. The polymerase photo-footprints in the near-consensus -35 hexamer on the non-template strand of DNA in a fashion similar to that of stable complexes involving the lacUV5 promoter. The kinetics of the interactions of polymerase alone with the tyrT promoter differ from those observed previously at the lacUV5 promoter. In the absence of FIS, we observe an upstream polymerase-induced signal at -122 within FIS site III that occurs subsequent to changes in the core promoter region and is strongly dependent on negative supercoiling. These observations support the proposal that the upstream region of the promoter is wrapped around the polymerase. We propose that the wrapped DNA allows the polymerase to overcome, at least in part, the barrier to DNA untwisting imparted by the G+C-rich discriminator. We further suggest that FIS plays a similar role and may facilitate polymerase escape.

Base Sequence↗
PubMed2001

The looped domain organization of the nucleoid in histone-like protein defective Escherichia coli strains.

We have investigated the major Escherichia coli histone-like proteins (H-NS, HU, FIS, and IHF) as putative factors involved in the maintenance of the overall DNA looped arrangement of the bacterial nucleoid. The long-range architecture of the chromosome has been studied by means of an assay based on in vivo genomic fragmentation mediated by endogenous DNA gyrase in the presence of oxolinic acid. The fragmentation products were analysed by CHEF electrophoresis. The results indicate that in vivo a large fraction of the bacterial chromatin constitutes an adequate substrate for the enzyme. DNA fragments released upon oxo-treatment span a size range from about 1000 kb to a limit-size of about 50 kb. The latter value is in excellent agreement with the average size reported for bacterial chromosomal domains. The DNA gyrase-mediated fragmentation does not appear to be significantly altered in strains depleted in histone-like proteins as compared to an E. coli wild type strain. This suggests that these proteins may not represent critical determinants for the maintenance of the supercoiled loop organisation of the E. coli chromosome.

Amino Acid Motifs↗
PubMed1992

Analysis of the Fis-dependent and Fis-independent transcription activation mechanisms of the Escherichia coli ribosomal RNA P1 promoter.

The role of the curved DNA sequence upstream to the Escherichia coli ribosomal RNA P1 promoter in transcription activation was studied. This sequence region had been shown to activate transcription from P1 in vivo and in vitro and to harbor binding sites for the trans-activating protein Fis. We have constructed a series of linker scanning mutants spanning the region -104 to -47, relative to the transcription start site. DNA fragments carrying the mutations show altered gel electrophoretic mobilities, consistent with reduced DNA bending angles compared to the wild-type sequence. Using gel retardation assays, qualitative as well as quantitative differences in the binding of the trans-activating protein Fis to the mutant DNA fragments could be observed. The effects of the mutations on rrnB P1 promoter activation were studied in vivo in fis+ and fis- backgrounds. A reduction in the promoter strength for some of the linker mutants correlates with altered Fis binding to two of the known Fis binding sites. Shifting the Fis binding region by half a helical turn, relative to the promoter core sequence, abolishes Fis-mediated activation almost totally, whereas activation is partly restored by a shift of a complete helical turn. For one mutant, which does not show alterations in Fis binding, a decrease in the promoter strength was observed in a fis- strain. From the results, we conclude that two upstream activating mechanisms, one Fis-dependent and one Fis-independent, influence the rrnB P1 promoter strength. Sequence determinants for the Fis-independent mechanism are closer to the promoter core region than the Fis binding sites. In addition, the study demonstrates that both the helical geometry and the absolute distance of the UAS region relative to the promoter are crucial for transcription activation.

Base Sequence↗
PubMedDate not supplied

Effects of FIS overexpression on cell growth, rRNA synthesis, and ribosome content in Escherichia coli.

The Escherichia coli DNA binding protein FIS is a transcriptional modulator involved in the regulation of many cellular processes, including the activation of rRNA synthesis. High-level overproduction of FIS in early, mid, or late log cultures resulted in growth-phase- and media-specific variations in cell growth, rRNA synthesis, and ribosome content. FIS overproduction caused a pronounced increase in rRNA synthesis for late-exponential cultures but a substantial reduction in cell growth and ribosome content. The addition of simple sugars such as glucose or fructose reversed these phenomena, consistent with the functional role of FIS in carbon metabolism.

Base Sequence↗
PubMedDate not supplied

Spatial relationship of the Fis binding sites for Hin recombinational enhancer activity.

Site-specific recombination reactions involve the joining or rearrangement of discrete DNA segments in a highly precise manner. A site-specific DNA inversion regulates the expression of flagellin genes in Salmonella by switching the orientation of a promoter. Analysis of the reaction has shown that, in addition to DNA sequences at the two boundaries of the 1-kilobase invertible segment where strand exchange occurs, another cis acting sequence is required for efficient inversion. This 60-base-pair enhancer-like sequence can function at many different locations and in either orientation in a plasmid substrate. It includes two binding sites for a host protein called Factor II or Fis (refs 4 and 5). Here we have investigated the importance of the spatial relationship between the two Fis binding sites for enhancer activity and have found that the correct helical positioning of the binding sites on the DNA is critical. However, this result could not be accounted for by effects on Fis binding. We propose a model for enhancer function in which the enhancer region acts to align the recombination sites into a specific conformation required for productive synapsis.

Bacterial Proteins↗
PubMed1997

FIS modulates growth phase-dependent topological transitions of DNA in Escherichia coli.

The Escherichia coli DNA-binding protein FIS serves as a DNA architectural factor in two unrelated enzymatic reactions, the site-specific inversion of DNA and transcriptional activation of stable RNA promoters. In both these processes, FIS facilitates the assembly and dynamic transitions of two structurally distinct nucleoprotein complexes. We have proposed previously that, in these systems, FIS stabilizes writhed DNA microloops by binding at multiple helically phased sites in DNA. However, FIS also binds and bends DNA at many non-specific sites and, at its maximum levels in the early exponential phase, FIS could potentially occupy a considerable part of the E. coli chromosome. Here, we show that fis affects growth phase-specific alterations in the supercoiling level of DNA. Expression of fis accelerates the accumulation of moderately supercoiled plasmids in stationary phase, which are stabilized by FIS after nutritional shift-up. In accordance with such a function, FIS modulates the relaxing and supercoiling activities of topoisomerases in vitro in a way that keeps DNA in a moderately supercoiled state. Our results suggest that the primary role of FIS is to modulate chromosomal dynamics during bacterial growth.

Carrier Proteins↗
PubMed2004

Escherichia coli prereplication complex assembly is regulated by dynamic interplay among Fis, IHF and DnaA.

Initiator DnaA and DNA bending proteins, Fis and IHF, comprise prereplication complexes (pre-RC) that unwind the Escherichia coli chromosome's origin of replication, oriC. Loss of either Fis or IHF perturbs synchronous initiation from oriC copies in rapidly growing E. coli. Based on dimethylsulphate (DMS) footprinting of purified proteins, we observed a dynamic interplay among Fis, IHF and DnaA on supercoiled oriC templates. Low levels of Fis inhibited oriC unwinding by blocking both IHF and DnaA binding to low affinity sites. As the concentration of DnaA was increased, Fis repression was relieved and IHF rapidly redistributed DnaA to all unfilled binding sites on oriC. This behaviour in vitro is analogous to observed assembly of pre-RC in synchronized E. coli. We propose that as new DnaA is synthesized in E. coli, opposing activities of Fis and IHF ensure an abrupt transition from a repressed complex with unfilled weak affinity DnaA binding sites to a completely loaded unwound complex, increasing both the precision of DNA replication timing and initiation synchrony.

Bacterial Proteins↗
PubMed1997

Molecular anatomy of a transcription activation patch: FIS-RNA polymerase interactions at the Escherichia coli rrnB P1 promoter.

FIS, a site-specific DNA binding and bending protein, is a global regulator of gene expression in Escherichia coli. The ribosomal RNA promoter rrnB P1 is activated 3- to 7-fold in vivo by a FIS dimer that binds a DNA site immediately upstream of the DNA binding site for the C-terminal domain (CTD) of the alpha subunit of RNA polymerase (RNAP). In this report, we identify several FIS side chains important specifically for activation of transcription at rrnB P1. These side chains map to positions 68, 71 and 74, in and flanking a surface-exposed loop adjacent to the helix-turn-helix DNA binding motif of the protein. We also present evidence suggesting that FIS activates transcription at rrnB P1 by interacting with the RNAP alphaCTD. Our results suggest a model for FIS-mediated activation of transcription at rrnB P1 that involves interactions between FIS and the RNAP alphaCTD near the DNA surface. Although FIS and the transcription activator protein CAP have little structural similarity, they both bend DNA, use a similarly disposed activation loop and target the same region of the RNAP alphaCTD, suggesting that this is a common architecture at bacterial promoters.

Arginine↗
PubMed2002

Transcriptional regulation of fis operon involves a module of multiple coupled promoters.

The transcription of the Escherichia coli fis gene is strongly activated during the outgrowth of cells from stationary phase. The high activity of the promoter of the fis operon requires the transcription factor IHF. Previously, we identified a divergent promoter, div, located upstream of the fis promoter. In this study we demonstrate that at least two additional promoters, designated fis P2 and fis P3, are located in the control region of the fis operon. The fis P2 and div promoters overlap completely, whereas fis P3 and div P are arranged as face-to-face divergent promoters. We show that the div and the tandem fis promoters counterbalance each other, such that their activity is kept on a lower than potentially attainable level. Furthermore, we demonstrate an unusual activation mechanism by IHF, involving a coordinated shift in the balance of promoter activities. We infer that these coupled promoters represent a regulatory module and propose a novel "dynamic balance" mechanism involved in the transcriptional control of the fis operon.

Base Sequence↗
PubMed1992

Both fis-dependent and factor-independent upstream activation of the rrnB P1 promoter are face of the helix dependent.

Transcription from the Escherichia coli rrnB P1 promoter is increased by a cis-acting sequence which extends upstream of the -35 hexamer to about -150 with respect to the transcription initiation site, the Upstream Activation Region (UAR). Activation by the UAR involves two components: (1) a trans-acting protein, Fis, which binds to three sites in the UAR between -60 and -150, and (2) the UAR sequences themselves which affect RNA polymerase (RNAP) activity independent of other proteins. We refer to the latter as Factor-Independent Activation (FIA). In addition to its interactions with the -10 and -35 hexamers typical of E. coli promoters, RNAP makes contacts to the -53 region of rrnB P1, which may be related to the FIA effect. We constructed a series of insertion mutants containing integral and non-integral numbers of helical turns at position -46, between the Fis binding sites and the -35 region, and the resulting promoter activities were measured in vitro and in vivo. The data suggest that both Fis-dependent and factor-independent activation are face of the helix dependent: the Fis binding site and the sequences responsible for factor-independent activation must be correctly oriented relative to RNA polymerase in order to activate transcription. These results, in conjunction with other evidence, support a model for the involvement of direct Fis-RNAP interactions in upstream activation. We also demonstrate that RNAP interacts with the -53 region of the rrnB P1 UAR even when these sequences are displaced upstream of the RNAP binding site, and that these interactions correlate with factor-independent activation.

Base Sequence↗
PubMed2001

An architectural role of the Escherichia coli chromatin protein FIS in organising DNA.

The Escherichia coli chromatin protein FIS modulates the topology of DNA in a growth phase-dependent manner. In this study we have investigated the global effect of FIS binding on DNA architecture in vitro. We show that in supercoiled DNA molecules FIS binds at multiple sites in a non-random fashion and increases DNA branching. This global DNA reshaping effect is independent of the helical phasing of FIS binding sites. We propose, in addition to the previously inferred stabilisation of tightly bent DNA microloops in the upstream regions of certain promoters, that FIS may perform the distinct architectural function of organising branched plectonemes in the E.coli nucleoid.

Carrier Proteins↗
PubMed1989

The Escherichia coli protein, Fis: specific binding to the ends of phage Mu DNA and modulation of phage growth.

We show, using gel retardation, that crude Escherichia coli cell extracts contain a protein which binds specifically to DNA fragments carrying either end of the phage Mu genome. We have identified this protein as Fis, a factor involved in several site-specific recombinational switches. Furthermore, we show that induction of a Mucts62 prophage in a fis lysogen occurs at a lower temperature than that of a wild-type strain, and that spontaneous induction of Mucts62 is increased in the fis mutant. DNasel footprinting using either crude extracts or purified Fis indicate that binding on the left end of Mu occurs at a site which overlaps a weak transposase binding site. Thus, Fis may modulate Mu growth by influencing the binding of transposase, or other proteins, to the transposase binding site(s), in a way similar to its influence on Xis binding in phage lambda.

Autoradiography↗
PubMed1993

Inhibition of bacteriophage Mu transposition by Mu repressor and Fis.

In this paper we show that the Escherichia coli protein Fis has a regulatory function in Mu transposition in the presence of Mu repressor. Fis can lower the transposition frequency of a mini-Mu 3-80-fold, but only if the Mu repressor is expressed simultaneously. In this novel type of regulation of transposition by the concerted action of Fis and repressor, the IAS, the internal activating sequence, is also involved as deletion of this site lead to the loss of the Fis effect. As the IAS contains strong repressor binding sites these are probably the target for the repressor in the observed negative regulation by Fis and repressor. However, the role of Fis and repressor is not only to inactivate the IAS, since a 4 bp insertion in the IAS, which changes the spacing of the repressor-binding site, abolishes the enhancing function of the IAS but leaves the repressor-Fis effect intact. A likely target for Fis in this regulation is a strong Fis-binding site, which is located adjacent to the L2 transposase-binding site. However, when this Fis-binding sequence was substituted by a random sequence and Fis no longer showed specific binding to this site, the Fis effect was still observed. Although it is still possible that Fis can function by binding to this non-specific site in a particular complex, it seems more likely that Fis is directly or indirectly involved in determining the level of the repressor.

Bacteriophage mu↗
PubMed1996

A positive control mutant of the transcription activator protein FIS.

The FIS protein is a transcription activator of rRNA and other genes in Escherichia coli. We have identified mutants of the FIS protein resulting in reduced rrnB P1 transcription activation that nevertheless retain the ability to bind DNA in vivo. The mutations map to amino acid 74, the N-terminal amino acid of the protein's helix-turn-helix DNA binding motif, and to amino acids 71 and 72 in the adjoining surface-exposed loop. In vitro analyses of one of the activation-defective mutants (with a G-to-S mutation at position 72) indicates that it binds to and bends rrnB P1 FIS site I DNA the same as wild-type FIS. These data suggest that amino acids in this region of FIS are required for transcription activation by contacting RNA polymerase directly, independent of any other role(s) this region may play in DNA binding or protein-induced bending.

Binding Sites↗
PubMed1998

In vivo assay of protein-protein interactions in Hin-mediated DNA inversion.

In order to form the catalytic nucleoprotein complex called the invertasome in the Hin-mediated DNA inversion reaction, interactions of the DNA-binding proteins Hin and Fis are required. Assays for these protein-protein interactions have been exploited with protein cross-linkers in vitro. In this study, an in vivo assay system that probes protein-protein interactions was developed. The formation of a DNA loop generated by protein interactions resulted in transcriptional repression of an artificially designed operon, which in turn increased the chance of survival of Escherichia coli host cells in a streptomycin-containing medium. Using this system, we were able to assay the Hin-Hin interaction that results in the pairing of the two recombination sites and protein interactions that result in the formation of the invertasome. This assay system also led us to find that an individual Hin dimer bound on a recombination site can form a stable complex with Fis bound on the recombinational enhancer; this finding has never been observed in in vitro studies. Possible pathways toward the formation of the invertasome are discussed based on the assay results for a previously reported Hin mutant.

Bacterial Proteins↗