Structural properties of the DNA‐bound form of a novel tandem repeat DNA‐binding domain, STPR

Saito, Shin; Yokoyama, Takuya; Aizawa, Tomoyasu; Kawaguchi, Kyosuke; Yamaki, Takeshi; Matsumoto, Daisuke; Kamijima, Tatsuro; Kamiya, Masakatsu; Kumaki, Yasuhiro; Mizuguchi, Mineyuki; Takiya, Shigeharu; Demura, Makoto; Kawano, Keiichi

doi:10.1002/prot.21939

Cited by 8 publications

(14 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The preparation of proteins without any tag was carried out as described (7). Crude extract was loaded onto a SP-Sepharose column, and fractionated with a linear gradient from 0 M to 1 M NaCl.…”

Section: Methodsmentioning

confidence: 99%

DNA-Binding Property of the Novel DNA-Binding Domain STPR in FMBP-1 of the Silkworm Bombyx mori

Takiya

Saito

Yokoyama

et al. 2009

Journal of Biochemistry

View full text Add to dashboard Cite

4 MATERIALS AND METHODSPreparation of FMBP-1 proteins -GST-fused FMBP-1 was prepared as described (5). For separation of the GST-tag from the GST-fused proteins, Sepharose beads bound with the fusion protein were washed with proteolysis buffer (50 mM Tris-HCl, pH7.0, 150 mM NaCl, 1 mM EDTA, and 1 mM DTT), and resuspended in a small amount of the same proteolysis buffer. PreScission protease (60 units/ml beads) was added and mixed with rotation overnight at 4 o C. The mixture was centrifuged, and the protein separated from GST in the supernatant was collected and dialyzed against NP40 buffer (50 mM Tris-HCl, pH7.9, 12.5 mM MgCl 2 , 100 mM NaCl, 0.1 mM EDTA, 20% glycerol, 0.1% NP40, and 1 mM DTT). Structures of the deletion proteins are described in Fig. 5A. POU-M1 was prepared with the same methods.The preparation of proteins without any tag was carried out as described (7). Crude extract was loaded onto a SP-Sepharose column, and fractionated with a linear gradient from 0 M to 1 M NaCl. The peak fractions were pooled and refractionated by reverse-phase HPLC.TFA was added to the eluate from the SP-Sepharose column, and the pH was adjusted to below 4.0. Samples were loaded on a reverse-phase HPLC column, and the proteins were eluted with a 0% -40% linear gradient of acetonitrile in 0.1% TFA. The peak fractions were pooled, lyophilized, redissolved in phosphate-buffered saline, and diluted with NP40 buffer.Electrophoretic Mobility shift assay -Electrophoretic mobility shift assays were carried out as described by Takiya et al. (4). The oligonucleotide probe +290 ( The FMBP-1-recognition sequence is composed of dA:dT pairs alone (ATNTWTNTA) (Fig.3), and all the minor groove-selecting reagents used here prefer AT-rich regions, whereas the major groove-selecting 7 reagent methyl green prefers GC-rich regions. Therefore, we examined effects of methyl green at up to 1 mM. As shown in Fig. 2B, 0.5 and 1 mM of methyl green tended to inhibit the binding of FMBP-1. These results suggested that FMBP-1 bound to the major groove of DNA.Distamycin gave an apparently controversial result (Fig. 2C).Distamycin binds selectively to the minor groove of DNA (10) We further analyzed the DNA-bending induced by these proteins with gels containing different concentrations of polyacrylamide. As shown in Fig. 5C, the angles calculated from the mobility in the different gels varied, but under all conditions used in this study, the STPR proteins with short extended regions did not bend DNA as sharply as the intact FMBP-1. However, we found that the STPR fused to GST at the N-terminus gave a similar pattern to the complete FMBP-1 in the permutation electrophoresis (Fig. 5D). Therefore, to bend DNA sharply, the long N-terminal extended region was necessary. A possible role of the long N-terminal extended region in the bending is discussed in the Discussion section. FMBP-1 can bind to DNA as a monomerThe leucine zipper-type transcription factors have a basic region on the N-terminal side of their DNA-binding domain, form homoand hetero-dimers,...

show abstract

Section: Methodsmentioning

confidence: 99%

DNA-Binding Property of the Novel DNA-Binding Domain STPR in FMBP-1 of the Silkworm Bombyx mori

Takiya

Saito

Yokoyama

et al. 2009

Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…Previous findings have suggested that the DNA-binding domains of TFs or DNA repair proteins might enter the groove of a DNA helix during the DNA sliding state; conformational changes in 3D structure are important for such interactions [32,35]. Findings from previous in vitro DNA-binding experiments involving STPR domains indicate that a conformational change into the a-helical-rich structure is important for stable DNA binding and that the R9A mutation abolishes this conformational change [13]. Thus, this conformational change might play an important role during 3rd component movements of the STPR as well as for stable DNA binding.…”

Section: Discussionmentioning

confidence: 99%

In vivo fluorescence correlation spectroscopy analyses of FMBP‐1, a silkworm transcription factor

et al. 2016

Self Cite

View full text Add to dashboard Cite

Fibroin modulator‐binding protein 1 ( FMBP ‐1) is a silkworm transcription factor that has a unique DNA ‐binding domain called the one score and three amino acid peptide repeat ( STPR ). Here we used fluorescence correlation spectroscopy ( FCS ) to analyze the diffusion properties of an enhanced green fluorescent protein‐tagged FMBP ‐1 protein ( EGFP ‐ FMBP ‐1) expressed in posterior silk gland ( PSG ) cells of Bombyx mori at the same developmental stage as natural FMBP ‐1 expression. EGFP ‐ FMBP ‐1 clearly localized to cell nuclei. From the FCS analyses, we identified an immobile DNA ‐bound component and three discernible diffusion components. We also used FCS to observe the movements of wild‐type and mutant EGFP ‐ FMBP ‐1 proteins in HeLa cells, a simpler experimental system. Based on previous in vitro observation, we also introduced a single amino acid substitution in order to suppress stable FMBP ‐1‐ DNA binding; specifically, we replaced the ninth Arg in the third repeat within the STPR domain with Ala. This mutation completely disrupted the slowest diffusion component as well as the immobile component. The diffusion properties of other FMBP ‐1 mutants (e.g. mutants with N‐terminal or C‐terminal truncations) were also analyzed. Based on our observations, we suggest that the four identifiable movements might correspond to four distinct FMBP ‐1 states: (a) diffusion of free protein, (b) and (c) two types of transient interactions between FMBP ‐1 and chromosomal DNA , and (d) stable binding of FMBP ‐1 to chromosomal DNA .

show abstract

“…Preparation of Recombinant STPR Peptides. Recombinant hSTPR and sSTPR peptides were prepared by an Escherichia coli expression system as described previously (13). The nucleotide sequence of the hSTPR domain was amplified using two oligonucleotide primers (5 0 -GGAATTCCATATGATGCGACAGA-ATGAGCCTTTGGAA-3 0 and 5 0 -CCGCTCGAGCATTTTC-TCCAGCCTCCTCTT-3 0 ) and the pOTB7 plasmid including cDNA of human ZNF821 (LOC55565) (purchased from Invitrogen, Carlsbad, CA).…”

Section: Methodsmentioning

confidence: 99%

STPR, a 23-Amino Acid Tandem Repeat Domain, Found in the Human Function-Unknown Protein ZNF821

et al. 2010

Self Cite

View full text Add to dashboard Cite

The STPR motif is composed of 23-amino acid repeats aligned contiguously. STPR was originally reported as the DNA-binding domain of the silkworm protein FMBP-1. ZNF821, the human protein that contains the STPR domain, is a zinc finger protein of unknown function. In this study, we prepared peptides of silkworm FMBP-1 STPR (sSTPR) and human ZNF821 STPR (hSTPR) and compared their DNA binding behaviors. This revealed that hSTPR, like sSTPR, is a double-stranded DNA-binding domain. Sequence-independent DNA binding affinities and α-helix-rich DNA-bound structures were comparable between the two STPRs, although the specific DNA sequence of hSTPR is still unclear. In addition, a subcellular expression experiment showed that the hSTPR domain is responsible for the nuclear localization of ZNF821. ZNF821 showed a much slower diffusion rate in the nucleus, suggesting the possibility of interaction with chromosomal DNA. STPR sequences are found in many proteins from vertebrates, insects, and nematodes. Some of the consensus amino acid residues would be responsible for DNA binding and concomitant increases in α-helix structure content.

show abstract

Structural properties of the DNA‐bound form of a novel tandem repeat DNA‐binding domain, STPR

Cited by 8 publications

References 35 publications

DNA-Binding Property of the Novel DNA-Binding Domain STPR in FMBP-1 of the Silkworm Bombyx mori

DNA-Binding Property of the Novel DNA-Binding Domain STPR in FMBP-1 of the Silkworm Bombyx mori

In vivo fluorescence correlation spectroscopy analyses of FMBP‐1, a silkworm transcription factor

STPR, a 23-Amino Acid Tandem Repeat Domain, Found in the Human Function-Unknown Protein ZNF821

Contact Info

Product

Resources

About