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The current focus of many functional genomic studies is on the elucidation of gene regulatory networks. The functional analyses of transcription factors and their DNA-binding sites, in conjunction with genome-wide expression profiling, are crucial in understanding of gene regulatory networks. This paper describes an efficient and easy method for characterizing the DNA-binding sequence specificity of novel plant transcription factors. This new method is based on the fusion of a DNA-binding protein (DBP) to 6×His-tagged cellulase D (CELD), which serves both as a means for affinity purification of DBP-DNA complex in the selection of binding sites from a pool of biotinylated random-sequence oligonucleotides and as a reporter for measurement of DNA-binding activity. Thus, it eliminates the use of radioactivity and gel electrophoresis techniques currently used for purification of DBP-DNA complexes and assays of DNA-binding activity. The effectiveness of this method was demonstrated by the success of simultaneous selection of the binding sites of nine plant DBPs from four superfamilies (AP2, bHLH, NAC and MYB). The high-throughput capacity of CELD-based DNA-binding assays allows the quantitative analysis of the binding sequence specificity from a large number of DBP-selected oligonucleotides. The binding sequence specificity of three novel transcription factors (rice OsbHLH66, wheat TaNAC69 and TaMYB80), determined with this method, is presented. This new method provides the capacity of high-throughput analysis on the DNA-binding sequence specificity of a large number of putative transcription factors, predicted on the basis of conserved DNA-binding domains. [ABSTRACT FROM AUTHOR]

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Salmonella enterica spp. produce siderophores to bind iron with high affinity and can also use three xenosiderophores secreted by other microorganisms, ferrichrome, coprogen, and ferrioxamine. Here we focused on FoxA, a TonB‐dependent transporter of ferrioxamines. Adjacent to foxA is a gene annotated as a helix‐turn‐helix (HTH) domain‐containing protein, SL0358 (foxR), in the Salmonella enterica serovar Typhimurium SL1344 genome. FoxR shares homology with transcriptional regulators belonging to the AraC/XylS family. foxR is syntenic with foxA in the Enterobacteriaceae family, suggesting their functional relatedness. Both foxA and foxR are repressed by the ferric uptake regulator (Fur) under iron‐rich growth conditions. When iron is scarce, FoxR acts as a transcriptional activator of foxA by directly binding to its upstream regulatory region. A point mutation in the HTH domain of FoxR abolished this binding, as did mutation of a direct repeat motif in the foxA upstream regulatory region. Desferrioxamine (DFOE) enhanced FoxR protein stability and foxA transcription but did not affect the affinity of FoxR binding to the foxA regulatory region. In summary, we have identified FoxR as a new member of the AraC/XylS family that regulates xenosiderophore‐mediated iron uptake by S. Typhimurium and likely other Enterobacteriaceae members. [ABSTRACT FROM AUTHOR]

Archaeal DNA recombination mechanism and the related proteins are similar to those in eukaryotes. However, no functional homolog of eukaryotic MutSγ, which recognizes Holliday junction to promote homologous recombination, has been identified in archaea. Hence, the whole molecular mechanism of archaeal homologous recombination has not yet been revealed. In this study, to identify the archaeal functional homolog of MutSγ, we focused on a functionally uncharacterized MutS homolog, MutS5, from a hyperthermophilic archaeon Pyrococcus horikoshii (phMutS5). Archaeal MutS5 has a Walker ATPase motif-containing amino acid sequence that shows similarity to the ATPase domain of MutSγ. It is known that the ATPase domain of MutS homologs is also a dimerization domain. Chemical cross-linking revealed that purified phMutS5 has an ability to dimerize in solution. phMutS5 bound to Holliday junction with a higher affinity than to other branched and linear DNAs, which resembles the DNA-binding specificities of MutSγ and bacterial MutS2, a Holliday junction-resolving MutS homolog. However, phMutS5 has no nuclease activity against branched DNA unlike MutS2. The ATPase activity of phMutS5 was significantly stimulated by the presence of Holliday junction similarly to MutSγ. Furthermore, site-directed mutagenesis revealed that the ATPase activity is dependent on the Walker ATPase motif of the protein. These results suggest that archaeal MutS5 should stabilize the Holliday junction and play a role in homologous recombination, which is analogous to the function of eukaryotic MutSγ. [ABSTRACT FROM AUTHOR]

Plastid envelope DNA-binding protein (PEND) is a DNA-binding protein with a chloroplast basic region-zipper domain at its N-terminus and a transmembrane domain at its C-terminus. The localization of PEND to the inner envelope membrane was demonstrated in a targeting experiment using isolated membranes and green fluorescent protein-tagged fusion proteins. An N-terminal sequence analysis showed that the presequence is 15 amino acids long; however, based on neural network-based prediction tools, this short peptide is not predicted to be a chloroplast-targeting sequence. In the present study we confirmed, by the digestion of intact chloroplasts, that PEND is located in the envelope membrane. We then demonstrated that the N-terminal 88-amino acid sequence is sufficient for plastid import in vitro. The transient expression of green fluorescent protein-tagged fusion proteins revealed that neither the N-terminal 29-amino acid sequence nor the 16-amino acid sequence directed green fluorescent protein to chloroplasts, but that the N-terminal 66-amino acid sequence was sufficient for correct targeting. These results suggest that targeting of PEND to the chloroplast requires both the presequence and the basic region, whereas postimport processing cleaves only the presequence. Interestingly, deletion of the presequence in the green fluorescent protein-tagged 88-amino acid construct resulted in targeting to the nucleus. This raises the possibility of plastid-to-nuclear signal transduction by the relocalization of PEND. [ABSTRACT FROM AUTHOR]

Sulfite reductase (SiR) is an important enzyme catalyzing the reduction of sulfite to sulfide during sulfur assimilation in plants. This enzyme is localized in plastids, including chloroplasts, and uses ferredoxin as an electron donor. Ferredoxin-dependent SiR has been found in isolated chloroplast nucleoids, but its localization in vivo or in intact plastids has not been examined. Here, we report the DNA-binding properties of SiRs from pea (PsSiR) and maize (ZmSiR) using an enzymatically active holoenzyme with prosthetic groups. PsSiR binds to both double-stranded and single-stranded DNA without significant sequence specificity. DNA binding did not affect the enzymatic activity of PsSiR, suggesting that ferredoxin and sulfite are accessible to SiR molecules within the nucleoids. Comparison of PsSiR and ZmSiR suggests that ZmSiR does indeed have DNA-binding activity, as was reported previously, but the DNA affinity and DNA-compacting ability are higher in PsSiR than in ZmSiR. The tight compaction of nucleoids by PsSiR led to severe repression of transcription activity in pea nucleoids. Indirect immunofluorescence microscopy showed that the majority of SiR molecules colocalized with nucleoids in pea chloroplasts, whereas no particular localization to nucleoids was detected in maize chloroplasts. These results suggest that SiR plays an essential role in compacting nucleoids in plastids, but that the extent of association of SiR with nucleoids varies among plant species. [ABSTRACT FROM AUTHOR]

Summary CDeT27-45 is a lea -like gene from the resurrection plant Craterostigma plantagineum (Scrophulariaceae) which is strongly expressed in vegetative tissues in response to dehydration or treatment with abscisic acid (ABA). Expression of the gene is correlated with the acquisition of desiccation tolerance. Nuclear proteins bind to a 29-bp cis -regulatory region of the promoter which is essential for transcriptional activation of the CDeT27-45 gene by ABA. Using a yeast one-hybrid screen, the cDNA clone CpR18 was isolated, which encodes a protein with specific binding activity for the cis -regulatory element in the CDeT27-45 promoter. The protein contains an acidic region, a SAP-domain, a zinc finger of the C3 H-type, and two motifs which are conserved in proteins from several plant species. One of the conserved regions is rich in basic residues and is predicted to form a helix-loop-helix structure. The R18 gene shows high similarities to genomic sequences and ESTs from other plant species. The tissue-specific expression pattern of the rare R18 mRNA and the distribution of nuclear protein binding activity for the CDeT27-45 promoter fragment are compared. The R18 protein is indeed localized in the nucleus, and activates transcription of CDeT27-45 promoter-GUS fusion constructs in tobacco protoplasts. DNA blot analysis and isolation of genomic clones reveal that two copies of R18 are present in the C. plantagineum genome. [ABSTRACT FROM AUTHOR]

In all organisms, type II DNA topoisomerases are essential for untangling chromosomal DNA. We have determined the structure of the DNA-binding core of the Methanococcusjannaschii DNA topoisomerase VIA subunit at 2.0 Å resolution. The overall structure of this subunit is unique, demonstrating that archaeal type II enzymes are distinct from other type II topoisomerases. However, the core structure contains a pair of domains that are also found in type IA and classic type II topoisomerases. Together, these regions may form the basis of a DNA cleavage mechanism shared among these enzymes. The core A subunit is a dimer that contains a deep groove that spans both protomers. The dimer architecture suggests that DNA is bound in the groove, across the A subunit interface, and that the two monomers separate during DNA transport. The A subunit of topoisomerase VI is homologous to the meiotic recombination factor, Spo11, and this structure can serve as a template for probing Spo11 function in eukaryotes. [ABSTRACT FROM AUTHOR]

The initiator protein (RepE) of F factor, a plasmid involved in sexual conjugation in Escherichia coli, has dual functions during the initiation of DNA replication which are determined by whether it exists as a dimer or as a monomer. A RepE monomer functions as a replication initiator, but a RepE dimer functions as an autogenous repressor. We have solved the crystal structure of the RepE monomer bound to an iteron DNA sequence of the replication origin of plasmid F. The RepE monomer consists of topologically similar N- and C-terminal domains related to each other by internal pseudo 2-fold symmetry, despite the lack of amino acid similarities between the domains. Both domains bind to the two major grooves of the iteron (19 bp) with different binding affinities. The C-terminal domain plays the leading role in this binding, while the N-terminal domain has an additional role in RepE dimerization. The structure also suggests that superhel- ical DNA induced at the origin of plasmid F by four RepEs and one HU dimer has an essential role in the initiation of DNA replication. [ABSTRACT FROM AUTHOR]