C.EcoO109I, a regulatory protein for production of EcoO109I restriction endonuclease, specifically binds to and bends DNA upstream of its translational start site

Kita, Keiko; Tsuda, Junko; Nakai, Shin‐ya

doi:10.1093/nar/gkf477

Cited by 32 publications

(34 citation statements)

References 24 publications

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“…The ORF therefore was designated the C.EcoT38I gene, which might produce a control protein for the EcoT38I R-M gene. Neither a conserved DNA sequence element termed a "C box," which is found immediately upstream of some C genes and is at least one target of C.PvuII binding (31), nor a C.EcoO109I-binding sequence (17) was found upstream of the C.EcoT38I gene translational start site. The GϩC contents of the R.EcoT38I, M.EcoT38I, and C.EcoT38I genes were 35, 42, and 38%, respectively.…”

Section: Resultsmentioning

confidence: 99%

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Evidence for Horizontal Transfer of the Eco T38I Restriction- Modification Gene to Chromosomal DNA by the P2 Phage and Diversity of Defective P2 Prophages in Escherichia coli TH38 Strains

2003

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A DNA fragment carrying the genes coding for a novel EcoT38I restriction endonuclease (R.EcoT38I) and EcoT38I methyltransferase (M.EcoT38I), which recognize G(A/G)GC(C/T)C, was cloned from the chromosomal DNA of Escherichia coli TH38. The endonuclease and methyltransferase genes were in a head-to-head orientation and were separated by a 330-nucleotide intergenic region. A third gene, the C.EcoT38I gene, was found in the intergenic region, partially overlapping the R.EcoT38I gene. The gene product, C.EcoT38I, acted as both a positive regulator of R.EcoT38I gene expression and a negative regulator of M.EcoT38I gene expression. M.EcoT38I purified from recombinant E. coli cells was shown to be a monomeric protein and to methylate the inner cytosines in the recognition sequence. R.EcoT38I was purified from E. coli HB101 expressing M.EcoT38I and formed a homodimer. The EcoT38I restriction (R)-modification (M) system (R-M system) was found to be inserted between the A and Q genes of defective bacteriophage P2, which was lysogenized in the chromosome at locI, one of the P2 phage attachment sites observed in both E. coli K-12 MG1655 and TH38 chromosomal DNAs. Ten strains of E. coli TH38 were examined for the presence of the EcoT38I R-M gene on the P2 prophage. Conventional PCR analysis and assaying of R activity demonstrated that all strains carried a single copy of the EcoT38I R-M gene and expressed R activity but that diversity of excision in the ogr, D, H, I, and J genes in the defective P2 prophage had arisen. Restriction (R)-modification (M) systems (R-M systems)serve as bacterial immune systems that destroy foreign DNA entering a cell. Typically, type II R-M systems consist of two enzymes: an endonuclease that recognizes and cleaves a specific DNA sequence and a methyltransferase that modifies the same sequence to protect the host chromosome from cleavage. More than 3,000 restriction endonucleases and methyltransferases have been isolated from many species of bacteria and have been subjected to biochemical and genetic studies (30). E. coli strains produce a variety of restriction endonucleases, including those of type I and III R-M systems. Type I and III systems are located on the chromosome; in contrast, most of the structural genes of type II R-M systems are located on a plasmid (5,20,27,38,39), and quite a few are located on chromosomal DNA (16, 21). Escherichia coli TH38, isolated from a pig, produces a type II restriction endonuclease, R.EcoT38I, that recognizes and cleaves the nucleotide sequence 5Ј-G(A/G)GC(C/T) 2C-3Ј (23). The occurrence of Hsd (host specificity for DNA) plasmids in E. coli TH38 has been investigated; one large plasmid was isolated from E. coli TH38, but Hsd ϩ transformants were not detected by use of a plasmid (38). These findings strongly suggest the possibility of chromosomal localization of the EcoT38I R-M system. Restriction endonucleases that show the same specificity as R.EcoT38I, such as R.BanII (14), have been isolated from a variety of bacteria, but none of the gene structures ha...

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Section: Resultsmentioning

confidence: 99%

“…The DNA fragment was excised with ApaI and SpeI and then ligated into ApaI/SpeIdigested pMCLTerAR (17). The activity of NADPH-dependent aldehyde reductase (AR1) (EC 1.1.1.2) at 37°C was determined by measuring the rate of decrease in the absorbance at 340 nm as described previously (17).…”

Section: Methodsmentioning

confidence: 99%

Evidence for Horizontal Transfer of the Eco T38I Restriction- Modification Gene to Chromosomal DNA by the P2 Phage and Diversity of Defective P2 Prophages in Escherichia coli TH38 Strains

2003

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“…Other groups, such as that exemplified by C.Csp231I and C.EcoO109I, have very different recognition sites and their structures are currently unknown. Kita et al (2002) have previously identified the recognition sequence of C.EcoO109I as a 15 bp sequence comprising two palindromic pentanucleotides separated by a nonbinding pentanucleotide sequence, 5 0 -CTAAG(N 5 )CTTAG-3 0 , located 47 bp upstream of the C gene start codon. This conforms to the sequence motif identified for both C.Csp231I and C.EcoO109I by bioinformatic analysis (Sorokin et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

Overexpression, purification and preliminary X-ray diffraction analysis of the controller protein C.Csp231I fromCitrobactersp. RFL231

2009

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Restriction-modification controller proteins play an essential role in regulating the temporal expression of restriction-modification genes. The controller protein C.Csp231I represents a new class of controller proteins. The gene was sublconed to allow overexpression in Escherichia coli. The protein was purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to 2.0 Å resolution and belonged to space group P2 1. An electrophoretic mobility-shift assay provided evidence of strong binding of C.Csp231I to a sequence located upstream of the csp231IC start codon. crystallization communications Acta Cryst. (2009). F65, 898-901 Streeter et al. C.Csp231i 901 Figure 5 Self-rotation function showing NCS. The self-rotation function is shown using a angle of 180 and was calculated using data in the resolution range 20.0-4.0 Å. The radius of integration was 35 Å .

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“…1. The C boxes have been shown in several cases, including PvuII (60), EcoRV (64), EcoO109I (27), and BamHI (J. Brooks, personal communication), to be bound by the cognate C protein. For PvuII, it has been shown that mutation of the C box can abolish this binding (60).…”

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confidence: 99%

Nature of the Promoter Activated by C.PvuII, an Unusual Regulatory Protein Conserved among Restriction-Modification Systems

et al. 2005

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A widely distributed family of small regulators, called C proteins, controls a subset of restriction-modification systems. The C proteins studied to date activate transcription of their own genes and that of downstream endonuclease genes; this arrangement appears to delay endonuclease expression relative to that of the protective methyltransferase when the genes enter a new cell. C proteins bind to conserved sequences called C boxes. In the PvuII system, the C boxes have been reported to extend from ؊23 to ؉3 relative to the transcription start for the gene for the C protein, an unexpected starting position relative to a bound activator. This study suggests that transcript initiation within the C boxes represents initial, C-independent transcription of pvuIICR. The major C protein-dependent transcript appears to be a leaderless mRNA starting farther downstream, at the initiation codon for the pvuIIC gene. This conclusion is based on nuclease S1 transcript mapping and the effects of a series of nested deletions in the promoter region. Furthermore, replacing the region upstream of the pvuIIC initiation codon with a library of random oligonucleotides, followed by selection for C-dependent transcription, yielded clones having sequences that resemble ؊10 promoter hexamers. The ؊35 hexamer of this promoter would lie within the C boxes. However, the spacing between C boxes/؊35 and the apparent ؊10 hexamer can be varied by ؎4 bp with little effect. This suggests that, like some other activator-dependent promoters, PpvuIICR may not require a ؊35 hexamer. Features of this transcription activation system suggest explanations for its broad host range.The genera hosting restriction-modification (RM) systems appear to comprise the majority of the prokaryotic world. RM systems play a variety of roles, ranging from the simply selfish (44, 54) through defense against bacteriophages (42, 51) to facilitating recombination (34, 36). While much remains to be learned about the functions of RM systems, our understanding of how they are regulated is even more limited. Most, if not all, RM system genes can move from cell to cell, either by residing on a plasmid or because of transduction, transformation, or Hfr-type conjugation. As a result, they all face a critical regulatory problem-how to ensure that a new host's DNA is protectively methylated before endonuclease activity appears. This problem is particularly acute for the type II RM systems, in which the methyltransferase (MTase) and endonuclease (REase) function independently.A subset of type II RM systems includes, in addition to genes for the MTase and REase, a third gene for a conserved regulator called the C (controller) protein. C proteins were originally discovered in the PvuII (58, 59) and BamHI (22, 57) RM systems and were then noted in several other systems as well

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C.EcoO109I, a regulatory protein for production of EcoO109I restriction endonuclease, specifically binds to and bends DNA upstream of its translational start site

Cited by 32 publications

References 24 publications

Evidence for Horizontal Transfer of the Eco T38I Restriction- Modification Gene to Chromosomal DNA by the P2 Phage and Diversity of Defective P2 Prophages in Escherichia coli TH38 Strains

Evidence for Horizontal Transfer of the Eco T38I Restriction- Modification Gene to Chromosomal DNA by the P2 Phage and Diversity of Defective P2 Prophages in Escherichia coli TH38 Strains

Overexpression, purification and preliminary X-ray diffraction analysis of the controller protein C.Csp231I fromCitrobactersp. RFL231

Nature of the Promoter Activated by C.PvuII, an Unusual Regulatory Protein Conserved among Restriction-Modification Systems

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