The Escherichia coli Fis protein prevents initiation of DNA replication from oriC in vitro

Wold, Sture; Crooke, Elliott; Skarstad, Kirsten

doi:10.1093/nar/24.18.3527

Cited by 61 publications

(59 citation statements)

References 38 publications

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“…Hence, this protein was termed factor for inversion stimulation (Fis). Subsequently, Fis was shown to be involved in other cellular processes, including bacteriophage DNA excision and integration (2, 3, 50), regulation of initiation of DNA replication at oriC (12,19,54), and regulation of gene expression (13). Fis can stimulate transcription of rRNA and tRNA operons (23,36,47) and several structural genes (1, 20, 56) and can also negatively regulate the transcription of various genes, including its own (3,20,39,55).…”

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confidence: 99%

Deletion analysis of the fis promoter region in Escherichia coli: antagonistic effects of integration host factor and Fis

et al. 1997

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Fis is a small DNA-binding and -bending protein in Escherichia coli that is involved in several different biological processes, including stimulation of specialized DNA recombination events and regulation of gene expression. fis protein and mRNA levels rapidly increase during early logarithmic growth phase in response to a nutritional upshift but become virtually undetectable during late logarithmic and stationary phases. We present evidence that the growth phase-dependent fis expression pattern is not determined by changes in mRNA stability, arguing in favor of regulation at the level of transcription. DNA deletion analysis of the fis promoter (fis P) region indicated that DNA sequences from ؊166 to ؊81, ؊36 to ؊26, and ؉107 to ؉366 relative to the transcription start site are required for maximum expression. A DNA sequence resembling the integration host factor (IHF) binding site centered approximately at ؊114 showed DNase I cleavage protection by IHF. In ihf cells, maximum cellular levels of fis mRNA were decreased more than 3-fold and transcription from fis P on a plasmid was decreased about 3.8-fold compared to those in cells expressing wild-type IHF. In addition, a mutation in the ihf binding site resulted in a 76 and 61% reduction in transcription from fis P on a plasmid in the presence or absence of Fis, respectively. Insertions of 5 or 10 bp between this ihf site and fis P suggest that IHF functions in a position-dependent manner. We conclude that IHF plays a role in stimulating transcription from fis P by interacting with a site centered approximately at ؊114 relative to the start of transcription. We also showed that although the fis P region contains six Fis binding sites, Fis site II (centered at ؊42) played a predominant role in autoregulation, Fis sites I and III (centered at ؉26 and ؊83, respectively) seemingly played smaller roles, and no role in negative autoregulation could be attributed to Fis sites IV, V, and VI (located upstream of site III). The fis P region from ؊36 to ؉7, which is not directly regulated by either IHF or Fis, retained the characteristic fis regulation pattern in response to a nutritional upshift.Fis is an 11.2-kDa DNA-binding and -bending protein that was first identified for its ability to stimulate DNA inversion reactions mediated by the Hin, Gin, and Cin family of recombinases (24, 29, 31). Hence, this protein was termed factor for inversion stimulation (Fis). Subsequently, Fis was shown to be involved in other cellular processes, including bacteriophage DNA excision and integration (2, 3, 50), regulation of initiation of DNA replication at oriC (12,19,54), and regulation of gene expression (13). Fis can stimulate transcription of rRNA and tRNA operons (23,36,47) and several structural genes (1, 20, 56) and can also negatively regulate the transcription of various genes, including its own (3,20,39,55). Roles played by Fis appear to be especially significant under conditions of rapid cell growth and high temperature, since fis cells grow slower than wild-type cells in r...

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confidence: 99%

Deletion analysis of the fis promoter region in Escherichia coli: antagonistic effects of integration host factor and Fis

et al. 1997

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“…The nature of these interactions is unknown. The introduction of a mutation in the primary Fis binding site in oriC does not overcome inhibition of in vitro replication (Wold et al, 1996). In contrast, our in vitro studies reveal that Fis in concert with IHF can promote the DnaA-mediated 13-mer melting in oriC (Fig.…”

Section: Discussionmentioning

confidence: 65%

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

Polaczek

Kwan

Liberles

et al. 1997

Molecular Microbiology

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SummaryBending 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 proteininduced bends, are critical cis-acting elements in specifying proper juxtapositioning of initiation factors in the early steps of DNA replication.

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“…1), and additional weaker binding sites for Fis have also been mapped within oriC (Polaczek, 1990;Filutowicz et al ., 1992). OriC plasmids carrying mutations that reduce binding of Fis and IHF have decreased function in vivo, although they can replicate in an in vitro assay (Roth et al ., 1994;Wold et al ., 1996). Strains lacking Fis or IHF also have perturbed initiation timing (Boye et al ., 1993).…”

Section: Orisome Componentsmentioning

confidence: 99%

“…Purified Fis inhibits in vitro open complex formation and replication of oriC plasmids (Hiasa and Marians, 1994;Wold et al ., 1996) by reducing binding of DnaA to lower affinity sites . Fis also inhibits IHF binding to oriC .…”

Section: Regulation Of Dnaa Loading Onto Oric -A Model For Assembly Omentioning

confidence: 99%

Building a bacterial orisome: emergence of new regulatory features for replication origin unwinding

Leonard

Grimwade

2005

Molecular Microbiology

107

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SummaryTriggering new rounds of chromosomal DNA replication during the bacterial cell cycle is exquisitely regulated, ensuring both proper timing and one round per cycle stringency. A critical first step is stable unwinding of oriC , the chromosomal replication origin, by multiprotein orisome complexes comprising the AAA + initiator DnaA and modulator proteins that bend DNA. Recently identified oriC -DnaA interactions in Escherichia coli raise important questions regarding the molecular mechanisms that regulate origin unwinding in bacteria. We describe staged binding of E. coli origin recognition proteins and suggest an unwinding switch based on interactions between DnaA-ATP and specialized oriC sites that must be filled during orisome assembly. By focusing multiple regulatory pathways on only a few key oriC DNA-protein interactions, this model includes an efficient way to control unwinding followed by orisome inactivation during the cell cycle. Future studies will determine whether this regulatory scheme is correct and whether it is generally applicable to other bacterial types.

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The Escherichia coli Fis protein prevents initiation of DNA replication from oriC in vitro

Cited by 61 publications

References 38 publications

Deletion analysis of the fis promoter region in Escherichia coli: antagonistic effects of integration host factor and Fis

Deletion analysis of the fis promoter region in Escherichia coli: antagonistic effects of integration host factor and Fis

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

Building a bacterial orisome: emergence of new regulatory features for replication origin unwinding

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