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1.
The ssh10b and ssh10b2 genes, a pair of distantly related paralogues in Sulfolobus shibatae, encode members of the Sac10b DNA binding protein family in thermophilic archaea. It has been shown previously that Ssh10b exists in abundance in S. shibatae and is capable of constraining negative DNA supercoils, properties that are consistent with a speculated architectural role for the protein in chromosomal organization. In this study, the ssh10b2 gene was cloned and expressed in Escherichia coli, and the recombinant Ssh10b2 protein was purified to apparent homogeneity. Immunoblotting analysis using a specific anti - Ssh10b2 antibody showed that ssh10b2 was expressed in S. shibatae, but the cellular level of Ssh10b2 was only - 10% of that of Ssh10b. Recombinant Ssh10b2 was capable of interacting with both double-stranded and single-stranded DNA. The affinity of the protein for double-stranded DNA was higher than that reported for Ssh10b. The Ssh10b2 and Ssh10b proteins appeared to generate similar gel shift patterns on duplex DNA fragments. However, unlike Ssh10b, Ssh10b2 was unable to constrain DNA supercoils. These data suggest that Ssh10b2 does not serve as a general architectural factor in DNA compaction and organization in S. shibatae.  相似文献   

2.
极端嗜热古菌———芝田硫化叶菌(Sulfolobus shibatae)基因组含一对亲缘关系较远的同源基因,ssh10b和ssh10b2。这对同源基因编码的蛋白(Ssh10b和Ssh10b2)属于古菌Sac10b DNA结合蛋白家族。关于Ssh10b以及与其极为相似的硫矿硫化叶菌(S.solfataricus)Sso10b、嗜酸热硫化叶菌(S.acidocaldarius)Sac10b蛋白已有较多研究,推测这些蛋白可能在染色体组织和包装、DNA重组、基因表达调控等方面起作用。克隆并在大肠杆菌中表达了ssh10b2基因,纯化了重组Ssh10b2蛋白。免疫印迹定量分析表明,ssh10b2在芝田硫化叶菌中有表达,但其细胞含量仅相当于Ssh10b的约十分之一。重组Ssh10b2对双链DNA的亲和力低于Ssh10b。此外,Ssh10b2和Ssh10b在与双链DNA结合时表现出相似的凝胶阻滞模式。有意思的是,Ssh10b2固定DNA负超螺旋的能力明显低于Ssh10b。这些结果提示,Ssh10b和Ssh10b2可能具有不同的生理作用。  相似文献   

3.
An 11.5-ku DNA binding protein, designated as Ssh12, was purified from the hyperthermophilic archaeonSulfolobus shibatae by column chromatography in SP Sepharose, DNA cellulose and phosphocellulose. Ssh12 accounts for about 4 % of the total cellular protein. The protein is capable of binding to both negatively supercoiled and relaxed DNAs. Nick closure analysis revealed that Ssh12 constrains negative supercoils upon binding to DNA. While the ability of the protein to constrain supercoils is weak at 22°C, it is enhanced substantially at temperatures higher than 37°C. Both the cellular content and supercoil-constraining ability of Ssh12 suggest that the protein may play an important role in the organization and stabilization of the chromosome ofS. shibatae.  相似文献   

4.
An 11.5-ku DNA binding protein, designated as Sshl2, was purified from the hyperthermophilic archaeon Sulfolobus shibatae by column chromatography in SP Sepharose, DNA cellulose and phosphocellulose. Sshl2 accounts for about 4 % of the total cellular protein. The protein is capable of binding to both negatively supercoiled and relaxed DNAs. Nick closure analysis revealed that Sshl2 constrains negative supercoils upon binding to DNA. While the ability of the protein to constrain supercoils is weak at 22℃ , it is enhanced substantially at temperatures higher than 37℃ . Both the cellular content and supercoil-constraining ability of Sshl2 suggest that the protein may play an important role in the organization and stabilization of the chromosome of S. shibatae.  相似文献   

5.
Fang X  Cui Q  Tong Y  Feng Y  Shan L  Huang L  Wang J 《Biochemistry》2008,47(43):11212-11221
The hyperthermophilic Ssh10b from Sulfolobus shibatae is a member of the Sac10b family, which has been postulated to play a role in chromosomal organization in Archaea. Ssh10b is capable of significantly constraining negative DNA supercoils at elevated temperatures. In this study, the solution structure of the dimeric P62A mutant Ssh10b ([P62A]Ssh10b) was determined by multidimensional NMR spectroscopy. The backbone 15N dynamics, H/D exchange with and without the denaturant GdmSCN, and chemical and thermal denaturation experiments were performed to investigate the molecular basis of high thermostability of [P62A]Ssh10b. Data analysis has revealed an alpha/beta-hydrophobic core consisting of two alpha-helices and one beta-sheet which are stabilized by cooperative hydrophobic and hydrogen-bonding interactions. This stabilizing alpha/beta-hydrophobic core of [P62A]Ssh10b exhibiting highly restricted internal motions is composed of residues having highly protected amide protons which exchange with solvent mostly by means of a global unfolding process. The K40N mutation greatly destabilizes the mutant [P62A]Ssh10b because this mutation disturbs the packing of alpha-helix against the beta-sheet reducing the stability of the alpha/beta-hydrophobic core in the mutant protein. In comparison with homologous mesophilic and thermophilic proteins, it can be presumed that the stabilizing alpha/beta-hydrophobic core in the [P62A]Ssh10b structure greatly contributes to the high thermostability of the protein.  相似文献   

6.
The DNA-binding protein Ssh10b from the hyperthermophilic archaeon Sulfolobus shibatae is a member of the Sac10b family, which has been speculated to be involved in the organization of the chromosomal DNA in Archaea. Ssh10b affects the DNA topology in a temperature dependent fashion that has not been reported for any other DNA-binding proteins. Heteronuclear NMR and site-directed mutagenesis were used to analyze the structural basis of the temperature-dependent Ssh10b-DNA interaction. The data analysis indicates that two forms of Ssh10b homodimers co-exist in solution, and the slow cis-trans isomerization of the Leu61-Pro62 peptide bond is the key factor responsible for the conformational heterogeneity of the Ssh10b homodimer. The T-form dimer, with the Leu61-Pro62 bond in the trans conformation, dominates at higher temperature, whereas population of the C-form dimer, with the bond in the cis conformation, increases on decreasing the temperature. The two forms of the Ssh10b dimer show the same DNA binding site but have different conformational features that are responsible for the temperature-dependent nature of the Ssh10b-DNA interaction.  相似文献   

7.
Guo R  Xue H  Huang L 《Molecular microbiology》2003,50(5):1605-1615
Proteins of the Sac10b family, which is highly conserved among hyperthermophilic archaea, have been regarded as DNA-binding proteins. Based on their in vitro DNA-binding properties, these proteins are thought to be involved in chromosomal organization or DNA repair/recombination. We show that Ssh10b, a member of the Sac10b family from Sulfolobus shibatae, bound with similar affinities to double-stranded DNA, single-stranded DNA and RNA in vitro. However, the protein was exclusively bound to RNA in S. shibatae cells, as revealed by in vivo UV cross-linking and co-immunoprecipitation. Ribosomal RNAs were among the RNA species co-immunoprecipitated with Ssh10b. Consistent with this observation, Ssh10b was co-purified with ribosomes under low salt conditions. Furthermore, we demonstrate by UV-cross-linking hybridization that, when the cells were irradiated with UV, Ssh10b became cross-linked to 16S, 23S rRNAs and mRNAs. Our data indicate that RNA is the physiological binding target of the Sac10b family.  相似文献   

8.
Major DNA binding proteins, designated Ssh7, were purified from the thermoacidophilic archaeon Sulfolobus shibatae. The Ssh7 proteins have an apparent molecular mass of 6.5 kDa and are similar to the 7-kDa DNA binding proteins from Sulfolobus acidocaldarius and Sulfolobus solfataricus in N-terminal amino acid sequence. The proteins constitute about 4.8% of the cellular protein. Upon binding to DNA, the Ssh7 proteins constrain negative supercoils. At the tested Ssh7/DNA mass ratios (0 to 1.65), one negative supercoil was taken up by approximately 20 Ssh7 molecules. Our results, together with the observation that the viral DNA isolated from S. shibatae is relaxed, suggest that regions of free DNA in the S. shibatae genome, if present, are highly positively supercoiled.  相似文献   

9.
极端嗜热古菌———芝田硫化叶菌 DNA 连接酶 (Ssh 连接酶 ) 的最适辅因子为 ATP ,在 dATP 存在时,该酶也能表现出较弱的连接活性 . ATP 或 dATP 都能够使该酶发生腺苷化,腺苷化的 Ssh 连接酶能够将腺苷基团转移至含切刻的 DNA 上 . 电泳迁移率改变实验表明, Ssh 连接酶能够结合双链 DNA ,且与含切刻及不含切刻的 DNA 结合的亲和力相同,但不结合单链 DNA. 酵母双杂交实验显示,硫磺矿硫化叶菌 ( 与芝田硫化叶菌亲缘关系很近 ) 的 DNA 连接酶,与该菌所含的 3 个增殖细胞核抗原 (PCNA) 同源蛋白中的一个 (PCNA-1) 有相互作用,而与另外 2 个同源蛋白 (PCNA-like 和 PCNA-2) 则无相互作用 . 在古菌中高度保守的 Sac10b 蛋白家族成员 Ssh10b 能够激活 Ssh 连接酶的活性,而硫化叶菌中的主要染色体蛋白——— 7 ku DNA 结合蛋白 (Ssh7) 则对该酶活性没有影响 .  相似文献   

10.
Liu YF  Zhang N  Liu X  Wang X  Wang ZX  Chen Y  Yao HW  Ge M  Pan XM 《PloS one》2012,7(4):e34986
The Sac10b protein family is regarded as a family of DNA-binding proteins that is highly conserved and widely distributed within the archaea. Sac10b family members are typically small basic dimeric proteins that bind to DNA with cooperativity and no sequence specificity and are capable of constraining DNA negative supercoils, protecting DNA from Dnase I digestion, and do not compact DNA obviously. However, a detailed understanding of the structural basis of the interaction of Sac10b family proteins with DNA is still lacking. Here, we determined the crystal structure of Mth10b, an atypical member of the Sac10b family from Methanobacterium thermoautotrophicum ΔH, at 2.2 Å. Unlike typical Sac10b family proteins, Mth10b is an acidic protein and binds to neither DNA nor RNA. The overall structure of Mth10b displays high similarity to its homologs, but three pairs of conserved positively charged residues located at the presumed DNA-binding surface are substituted by non-charged residues in Mth10b. Through amino acids interchanges, the DNA-binding ability of Mth10b was restored successfully, whereas the DNA-binding ability of Sso10b, a typical Sac10b family member, was weakened greatly. Based on these results, we propose a model describing the molecular mechanism underlying the interactions of typical Sac10b family proteins with DNA that explains all the characteristics of the interactions between typical Sac10b family members and DNA.  相似文献   

11.
极端嗜热古菌--芝田硫化叶菌DNA结合蛋白Ssh7a和Ssh7b的编码基因(ss7α和ssh7ь)在大肠杆菌中得到表达。量均达到细胞蛋白总量的10% ̄15%。重组蛋白通过一个包括热处理步骤的简单纯化程序得到纯化。重组Ssh7a和Ssh7b与松弛及负超螺旋DNA的结合与天然Ssh7蛋白无异,与天然Ssh7相似,Ssh7a在与DNA结合时能免固定负超螺旋,每固定一个负超螺旋约需22个Ssh7a分子。这  相似文献   

12.
Proteins of the Sac10b family are highly conserved in Archaea. Ssh10b, a member of the Sac10b family from the hyperthermophilic crenarchaeon Sulfolobus shibatae, binds to RNA in vivo. Here we show that binding by Ssh10b destabilizes RNA secondary structure. Structural analysis of Ssh10b in complex with a 25-bp RNA duplex containing local distortions reveals that Ssh10b binds the two RNA strands symmetrically as a tetramer with each dimer bound asymmetrically to a single RNA strand. Amino acid residues involved in double-stranded RNA binding are similar, but non-identical, to those in dsDNA binding. The dimer-dimer interaction mediated by the intermolecular β-sheet appears to facilitate the destabilization of base pairing in the secondary structure of RNA. Our results suggest that proteins of the Sac10b family may play important roles in RNA transactions requiring destabilization of RNA secondary structure in Sulfolobus.  相似文献   

13.
The α/β-mixed dimeric protein Ssh10b from the hyperthermophile Sulfolobus shibatae is a member of the Sac10b family that is thought to be involved in chromosomal organization or DNA repair/recombination. The equilibrium unfolding/refolding of Ssh10b induced by denaturants and heat was fully reversible, suggesting that Ssh10b could serve as a good model for folding/unfolding studies of protein dimers. Here, we investigate the folding/unfolding kinetics of Ssh10b in detail by stopped-flow circular dichroism (SF-CD) and using GdnHCl as denaturant. In unfolding reactions, the native Ssh10b turned rapidly into fully unfolded monomers within the stopped-flow dead time with no detectable kinetic intermediate, agreeing well with the results of equilibrium unfolding experiments. In refolding reactions, two unfolded monomers associate in the burst phase to form a dimeric intermediate that undergoes a further, slower, first-order folding process to form the native dimer. Our results demonstrate that the dimerization is essential for maintaining the native tertiary interactions of the protein Ssh10b. In addition, folding mechanisms of Ssh10b and several other α/β-mixed or pure β-sheet proteins are compared.  相似文献   

14.
Small-angle X-ray scattering has been used to study the structure of the multimeric complexes that form between double-stranded DNA and the archaeal chromatin protein Sac7d from Sulfolobus acidocaldarius. Scattering data from complexes of Sac7d with a defined 32-mer oligonucleotide, with poly[d(GC)], and with E. coli DNA indicate that the protein binds along the surface of an extended DNA structure. Molecular models of fully saturated Sac7d/DNA complexes were constructed using constraints from crystal structure and solution binding data. Conformational space was searched systematically by varying the parameters of the models within the constrained set to find the best fits between the X-ray scattering data and simulated scattering curves. The best fits were obtained for models composed of repeating segments of B-DNA with sharp kinks at contiguous protein binding sites. The results are consistent with extrapolation of the X-ray crystal structure of a 1:1 Sac7d/octanucleotide complex [Robinson, H., et al. (1998) Nature 392, 202-205] to polymeric DNA. The DNA conformation in our multimeric Sac7d/DNA model has the base pairs tilted by about 35 degrees and displaced 3 A from the helix axis. There is a large roll between two base pairs at the protein-induced kink site, resulting in an overall bending angle of about 70 degrees for Sac7d binding. Regularly repeating bends in the fully saturated complex result in a zigzag structure with negligible compaction of DNA. The Sac7d molecules in the model form a unique structure with two left-handed helical ribbons winding around the outside of the right-handed duplex DNA.  相似文献   

15.
The thermoacidophilic archaeon Sulfolobus shibatae synthesizes a large amount of the 7-ku DMA binding proteins known as Ssh7. Our hybridization experiments showed that two Ssh7-encoding genes existed in the genome of S. shibatae. These two genes, designated ssh7a and ssh7b, have been cloned, sequenced and expressed in Escherichia coli. The two Ssh7 proteins differ only at three amino acid positions. In addition, the cis-regulatory sequences of the ssh7a and ssh7b genes are highly conserved. These results suggest the presence of a selective pressure to maintain not only the sequence but also the expression of the two genes. We have also found that there are two genes encoding the 7-ku protein in Sulfolobus solfataricus. Based on this and other studies, we suggest that the gene encoding the 7-ku protein underwent duplication before the separation of Sulfolobus species. Binding of native Ssh7 and recombinant (r)Ssh7 to short duplex DNA fragments was analyzed by electrophoretic mobility shift assays. Both n  相似文献   

16.
Dostál L  Chen CY  Wang AH  Welfle H 《Biochemistry》2004,43(30):9600-9609
Members of the Sso7d/Sac7d protein family and other related proteins are believed to play an important role in DNA packaging and maintenance in archeons. Sso7d/Sac7d are small, abundant, basic, and nonspecific DNA-binding proteins of the hyperthermophilic archeon Sulfolobus. Structures of several complexes of Sso7d/Sac7d with DNA octamers are known. These structures are characterized by sequence unspecific minor groove binding of the proteins and sharp kinking of the double helix. Corresponding Raman vibrational signatures have been identified in this study. A Raman spectroscopic analysis of Sac7d binding to the oligonucleotide decamer d(GAGGCGCCTC)(2) reveals large conformational perturbations in the DNA structure upon complex formation. Perturbed Raman bands are associated with the vibrational modes of the sugar phosphate backbone and frequency shifts of bands assigned to nucleoside vibrations. Large changes in the DNA backbone and partial B- to A-form DNA transitions are indicated that are closely associated with C2'-endo/anti to C3'-endo/anti conversion of the deoxyadenosyl moiety upon Sac7d binding. The major spectral feature of Sac7d binding is kinking of the DNA. Raman markers of minor groove binding do not largely contribute to spectral differences; however, clear indications for minor groove binding come from G-N2 and G-N3 signals that are supported by Trp24 features. Trp24 is the only tryptophan present in Sac7d and binds to guanine N3, as has been demonstrated clearly in X-ray structures of Sac7d-DNA complexes. No changes of the Sac7d secondary structure have been detected upon DNA binding.  相似文献   

17.
Sac7d is a hyperthermophile chromatin protein which binds non-specifically to the minor groove of duplex DNA and induces a sharp kink of 66 degrees with intercalation of valine and methionine side-chains. We have utilized the thermal stability of Sac7d and the lack of sequence specificity to define the thermodynamics of DNA binding over a wide temperature range. The binding affinity for poly(dGdC) was moderate at 25 degrees C (Ka = 3.5(+/-1.6) x 10(6) M(-1)) and increased by nearly an order of magnitude from 10 degrees C to 80 degrees C. The enthalpy of binding was unfavorable at 25 degrees C, and decreased linearly from 5 degrees C to 60 degrees C. A positive binding heat at 25 degrees C is attributed in part to the energy of distorting DNA, and ensures that the temperature of maximal binding affinity (75.1+/-5.6 degrees C) is near the growth temperature of Sulfolobus acidocaldarius. Truncation of the two intercalating residues to alanine led to a decreased ability to bend and unwind DNA at 25 degrees C with a small decrease in binding affinity. The energy gained from intercalation is slightly greater than the free energy penalty of bending duplex DNA. Surprisingly, reduced distortion from the double alanine substitution did not lead to a significant decrease in the heat of binding at 25 degrees C. In addition, an anomalous positive DeltaCp of binding was observed for the double alanine mutant protein which could not be explained by the change in polar and apolar accessible surface areas. Both the larger than expected binding enthalpy and the positive heat capacity can be explained by a temperature dependent structural transition in the protein-DNA complex with a Tm of 15-20 degrees C and a DeltaH of 15 kcal/mol. Data are discussed which indicate that the endothermic transition in the complex is consistent with DNA distortion.  相似文献   

18.
Cren7, a newly found chromatin protein, is highly conserved in the Crenarchaeota. The protein shows higher affinity for double‐stranded DNA than for single‐stranded DNA, constrains negative DNA supercoils in vitro and is associated with genomic DNA in vivo. Here we report the crystal structures of the Cren7 protein from Sulfolobus solfataricus in complex with two DNA sequences. Cren7 binds in the minor groove of DNA and causes a single‐step sharp kink in DNA (~53°) through the intercalation of the hydrophobic side chain of Leu28. Loop β3‐β4 of Cren7 undergoes a significant conformational change upon binding of the protein to DNA, suggesting its critical role in the stabilization of the protein–DNA complex. The roles of DNA‐contacting amino acid residues in stabilizing the Cren7–DNA interaction were examined by mutational analysis. Structural comparison of Cren7‐DNA complexes with Sac7d‐DNA complexes reveals significant differences between the two proteins in DNA binding surface, suggesting that Cren7 and Sul7d serve distinct functions in chromosomal organization.  相似文献   

19.
Mvo10b from the mesophilic archaeon Methanococcus voltae is a member of the Sac10b family which may play an important role in the organization and accessibility of genetic information in Archaea. Since Mvo10b is a DNA-binding protein as the other member in the Sac10b family, to obtain a recombinant Mvo10b requires an efficient and inexpensive expression and purification system for producing the protein free of nucleic acid contamination. Previously, the hyperthermophilic archaeal Ssh10b of the Sac10b family was successfully purified. However, the protocol adopted to purify Ssh10b is not appropriate for purifying the mesophilic Mvo10b. This study describes the successful expression and purification of the recombinant Mvo10b. The expression of recombinant Mvo10b was carried out in Escherichia coli, and the target protein was expressed in the soluble form. The protein was purified by polyethyleneimine (PEI) precipitation followed by nickel ion metal affinity chromatography. The purity of Mvo10b was checked to insure being free of nucleic acid contamination. The final protein yield is about 30 mg/l of LB culture. The ensemble of NMR and far-UV CD data shows that the purified Mvo10b has abundant regular secondary structures and is correctly folded, which may have similar 3D structure as its hyperthermophilic counterpart [P62A]Ssh10b. The developed protocol has potential application in the production of the other thermophilic and mesophilic proteins in the Sac10b family.  相似文献   

20.
Sac7d is a small chromatin protein from the hyperthermophile Sulfolobus acidocaldarius which kinks duplex DNA by approximately 66 degrees at a single base pair step with intercalation of V26 and M29 side chains. Site-directed mutagenesis coupled with calorimetric and spectroscopic data has been used to characterize the influence of the intercalating side chains on the structure and thermodynamics of the DNA complex from 5 to 85 degrees C. Two single-alanine substitutions (V26A and M29A) and five double-glycine, -alanine, -leucine, -phenylalanine, and -tryptophan substitutions of the surface residues have been created. NMR and fluorescence titrations indicated that the substitutions had little effect on the structure of the protein or DNA binding site size. Each of the mutant proteins demonstrated a temperature-dependent binding enthalpy which was correlated with a similar temperature dependence in the structure of the complex reflected by changes in fluorescence and circular dichroism. A positive heat capacity change (DeltaC(p)) for DNA binding was observed for only those mutants which also demonstrated a thermotropic structural transition in the complex, and the temperature range for the positive DeltaC(p) coincided with that observed for the structural transition. The thermodynamic data are interpreted using a model in which binding is linked to an endothermic distortion of the DNA in the complex. The results support the proposal that the unfavorable enthalpy of binding of Sac7d at 25 degrees C is due in part to the distortion of DNA.  相似文献   

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