Interactions of Archaeal Chromatin Proteins Alba1 and Alba2 with Nucleic Acids

Background

Architectural proteins have important roles in compacting and organising chromosomal DNA. There are two potential histone counterpart peptide sequences (Alba1 and Alba2) in the Aeropyrum pernix genome (APE1832.1 and APE1823).

Methodology/Principal Findings

These two peptides were expressed and their interactions with various DNAs were studied using a combination of various experimental techniques: surface plasmon resonance, UV spectrophotometry, circular dichroism–spectropolarimetry, gel-shift assays, and isothermal titration calorimetry.

Conclusions/Significance

Our data indicate that there are significant differences in the properties of the Alba1 and Alba2 proteins. Both of these Alba proteins can thermally stabilise DNA polynucleotides, as seen from UV melting curves. Alba2 and equimolar mixtures of Alba1/Alba2 have greater effects on the thermal stability of poly(dA-dT).poly(dA-dT). Surface plasmon resonance sensorgrams for binding of Alba1, Alba2, and equimolar mixtures of Alba1/Alba2 to DNA oligonucleotides show different binding patterns. Circular dichroism indicates that Alba2 has a less-ordered secondary structure than Alba1. The secondary structures of the Alba proteins are not significantly influenced by DNA binding, even at high temperatures. Based on these data, we conclude that Alba1, Alba2, and equimolar mixtures of Alba1/Alba2 show different properties in their binding to various DNAs.     

Sir2 and the acetyltransferase, Pat, regulate the archaeal chromatin protein, Alba

The DNA binding affinity of Alba, a chromatin protein of the archaeon Sulfolobus solfataricus P2, is regulated by acetylation of lysine 16. Here we identify an acetyltransferase that specifically acetylates Alba on this residue. The effect of acetylation is to lower the affinity of Alba for DNA. Remarkably, the acetyltransferase is conserved not only in archaea but also in bacteria where it appears to play a role in metabolic regulation. Therefore, our data suggest that S. solfataricus has co-opted this bacterial regulatory system to generate a rudimentary form of chromatin regulation.     

Crystal structure of archaeal chromatin protein Alba2-double-stranded DNA complex from Aeropyrum pernix K1

All thermophilic and hyperthermophilic archaea encode homologs of dimeric Alba (Sac10b) proteins that bind cooperatively at high density to DNA. Here, we report the 2.0 Å resolution crystal structure of an Alba2 (Ape10b2)-dsDNA complex from Aeropyrum pernix K1. A rectangular tube-like structure encompassing duplex DNA reveals the positively charged residues in the monomer-monomer interface of each dimer packing on either side of the bound dsDNA in successive minor grooves. The extended hairpin loop connecting strands β3 and β4 undergoes significant conformational changes upon DNA binding to accommodate the other Alba2 dimer during oligomerization. Mutational analysis of key interacting residues confirmed the specificity of Alba2-dsDNA interactions.     

Archaeal chromatin proteins histone HMtB and Alba have lost DNA-binding ability in laboratory strains of <Emphasis Type="Italic">Methanothermobacter thermautotrophicus</Emphasis>

Alignments of the sequences of the all members of the archaeal histone and Alba1 families of chromatin proteins identified isoleucine residues, I19 in HMtB and I39 in MtAlba, in Methanothermobacter thermautotrophicus, at locations predicted to be directly involved in DNA binding. In all other HMfB family members, residue 19 is an arginine (R19), and either arginine or lysine is present in almost all other Alba1 family members at the structural site equivalent to I39 in MtAlba. Electrophoretic mobility shift assays revealed that recombinant HMtB and MtAlba do not bind DNA, but variants constructed with R19 and R39, respectively, bound DNA; and whereas MtAlba(I19) did not bind RNA, MtAlba(R19) bound both single stranded RNA and tRNA. Amplification and sequencing of MT0254 (encodes HMtB) and MT1483 (encodes MtAlba) from several Methanothermobacter thermautotrophicus lineages has revealed that HMtB and MtAlba had arginine residues at positions 19 and 39, respectively, in the original isolate and that spontaneous mutations must have occurred, and been fixed, in some laboratory lineages that now have HMtB(I19) and MtAlba(I39). The retention of these variants suggests some continuing functions and fusion of the HMtB(I19) sequence to HMtA2 resulted in a protein that folds to form a histone fold heterodimer that binds and compacts DNA. The loss of DNA binding by HMtB(I19) does not therefore prevent HMtB from participating in DNA interactions as one partner of an archaeal histone heterodimer.     

CC1, a novel crenarchaeal DNA binding protein

The genomes of the related crenarchaea Pyrobaculum aerophilum and Thermoproteus tenax lack any obvious gene encoding a single-stranded DNA binding protein (SSB). SSBs are essential for DNA replication, recombination, and repair and are found in all other genomes across the three domains of life. These two archaeal genomes also have only one identifiable gene encoding a chromatin protein (the Alba protein), while most other archaea have at least two different abundant chromatin proteins. We performed a biochemical screen for novel nucleic acid binding proteins present in cell extracts of T. tenax. An assay for proteins capable of binding to a single-stranded DNA oligonucleotide resulted in identification of three proteins. The first protein, Alba, has been shown previously to bind single-stranded DNA as well as duplex DNA. The two other proteins, which we designated CC1 (for crenarchaeal chromatin protein 1), are very closely related to one another, and homologs are restricted to the P. aerophilum and Aeropyrum pernix genomes. CC1 is a 6-kDa, monomeric, basic protein that is expressed at a high level in T. tenax. This protein binds single- and double-stranded DNAs with similar affinities. These properties are consistent with a role for CC1 as a crenarchaeal chromatin protein.     

Crystal structure of an archaeal specific DNA-binding protein (Ape10b2) from Aeropyrum pernix K1

DNA binding proteins are essential in all organisms, and they play important roles in both compacting and regulating the genetic material. All thermophilic and hyperthermophilic archaea encode one or more copies of Alba or Sso10b, which is a small, abundant, basic protein that binds DNA. Here, we present the crystal structure of Ape10b2 from Aeropyrum pernix K1 at 1.70 A. Although the overall structure resembles the known Alba protein fold, a significant conformational change was observed in the loop regions. Specifically, the L5 loop is slightly longer, as compared to those of other known proteins, and the flexibility of this loop may facilitate the interaction with double stranded DNA. In addition, we showed that Ape10b2 binds to 16 and 39 bp duplex DNAs with high affinity. On the basis of our analyses, we have created a putative protein-DNA complex model.     

The two faces of Alba: the evolutionary connection between proteins participating in chromatin structure and RNA metabolism

Background  

There is considerable heterogeneity in the phyletic patterns of major chromosomal DNA-binding proteins in archaea. Alba is a well-characterized chromosomal protein from the crenarchaeal genus Sulfolobus. While Alba has been detected in most archaea and some eukaryotic taxa, its exact functions in these taxa are not clear. Here we use comparative genomics and sequence profile analysis to predict potential alternative functions of the Alba proteins.     

高速逆流色谱法从白芍中分离纯化五没食子酰基葡萄糖

本文建立高速逆流色谱(HSCCC)方法,从白芍粗提物中分离纯化五没食子酰基葡萄糖.分别采用正己烷-乙酸乙酯-甲醇-水体积比0.5∶5∶1∶5及0.5∶5∶0.5∶5混合溶剂作为两相溶剂体系,上相为固定相,下相为流动相,转速为800 rpm,流速为2.0 mL/min,用HPLC检测及ESI-MS进行验证.经过两次HSCCC分离纯化,得到五没食子酰基葡萄糖纯度为95.7％.     

Advances in finding Alba: the locus affecting life history and color polymorphism in a Colias butterfly

Although alternative life‐history strategies exist within many populations, very little is known about their genetic basis and mechanistic insight into these traits could greatly advance the understanding of eco‐evolutionary dynamics. Many species of butterfly within the genus Colias exhibit a sex‐limited wing colour polymorphism, called Alba, which is correlated with an alternative life‐history strategy. Here, we have taken the first steps in localizing the region carrying Alba in Colias croceus, a species with no genomic resources, by generating whole genome sequence of a single Alba mother and two sequencing pools, one for her Alba and another for her orange, offspring. These data were used in a bulk‐segregant analysis wherein SNPs fulfilling the Mendelian inheritance expectations of Alba were identified. Then, using the conserved synteny in Lepidoptera, the Alba locus was assigned to chromosome 15 in Bombyx mori. We then identified candidate regions within the chromosome by investigating the distribution of Alba SNPs along the chromosome and the difference in nucleotide diversity in exons between the two pools. A region spanning ~ 5.7 Mbp at the 5′ end of the chromosome was identified as likely to contain the Alba locus. These insights set the stage for more detailed genomic scans and mapping of the Alba phenotype, and demonstrate an efficient use of genomic resources in a novel species.     

Immunoregulation and antioxidant activities of a novel acidic polysaccharide from Radix Paeoniae Alba

Radix Paeoniae Alba is widely used in Chinese traditional medicine to treat various diseases such as gastrointestinal disorders, immunomodulatory, cancer, and other diseases. In this paper, a novel acidic polysaccharide RPAPS purified from Radix Paeoniae Alba was evaluated for its structural features and potential of immunomodulatory and antioxidant activities. RPAPS (molecular weight: 1.0× 105 Da) was mainly composed of α-(1 → 4)-Glcp, α-Arap, α-Galp, α-Rhap, β-D-Glcp, α-(1 → 6)-linked Glcp and GalA. Immunological tests indicated that RPAPS could improve RAW264.7 phagocytic activity and LPS-induced splenocyte proliferation. For antioxidant activities, RPAPS showed reducing power and DPPH scavenging activity in dose dependent. Moreover, RPAPS could significantly protect the PC12 cells from H2O2 damage. These data implied polysaccharides RPAPS had the potential to be novel natural antioxidative and immunopotentiating agents for using in functional foods or medicine.     

Crystal Structure of Human Rpp20/Rpp25 Reveals Quaternary Level Adaptation of the Alba Scaffold as Structural Basis for Single-stranded RNA Binding

Ribonuclease P (RNase P) catalyzes the removal of 5′ leaders of tRNA precursors and its central catalytic RNA subunit is highly conserved across all domains of life. In eukaryotes, RNase P and RNase MRP, a closely related ribonucleoprotein enzyme, share several of the same protein subunits, contain a similar catalytic RNA core, and exhibit structural features that do not exist in their bacterial or archaeal counterparts. A unique feature of eukaryotic RNase P/MRP is the presence of two relatively long and unpaired internal loops within the P3 region of their RNA subunit bound by a heterodimeric protein complex, Rpp20/Rpp25. Here we present a crystal structure of the human Rpp20/Rpp25 heterodimer and we propose, using comparative structural analyses, that the evolutionary divergence of the single-stranded and helical nucleic acid binding specificities of eukaryotic Rpp20/Rpp25 and their related archaeal Alba chromatin protein dimers, respectively, originate primarily from quaternary level differences observed in their heterodimerization interface. Our work provides structural insights into how the archaeal Alba protein scaffold was adapted evolutionarily for incorporation into several functionally-independent eukaryotic ribonucleoprotein complexes.     

Crystal structure and functional analysis of an archaeal chromatin protein Alba from the hyperthermophilic archaeon Pyrococcus horikoshii OT3

The crystal structure of the Alba protein (PhoAlba) from a hyperthermophilic archaeon, Pyrococcus horikoshii OT3, was determined at a resolution of 2.8 A. PhoAlba structurally belongs to the alpha/beta proteins and is similar not only to archaeal homologues but also to RNA-binding proteins, including the C-terminal half of initiation factor 3 (IF3-C) from Bacillus stearothermophilus, an Esherichia coli protein implicated in cell division (Yhhp), and an Arabidopsis protein of unknown function. We found by gel shift assay that PhoAlba interacts with both ribonuclease P (RNase P) RNA (PhopRNA) and precursor-tRNA(Tyr) (pre-tRNA(Tyr)) in P. horikoshii. However, the addition of PhoAlba to reconstituted particles composed of PhopRNA and four or five protein subunits had little influence on either the pre-tRNA processing activity or the optimum temperature for the processing activity. These results suggest that PhoAlba contributes little to the catalytic activity of P. horikoshii RNase P.     

HPLC特征图谱结合含量测定的白芍及其炮制品的质量对比研究

建立白芍、炒白芍、酒白芍、硫熏白芍HPLC特征图谱,并结合多成分含量测定,为白芍、炒白芍、酒白芍和硫熏白芍的质量控制提供参考。采用Intersustain C₁₈(250 mm×4.6 mm,5μm)色谱柱,流动相为乙腈-0.1%醋酸水溶液,流速为每分钟1 mL,梯度洗脱,检测波长为230 nm,柱温为30℃,进样量为10μL。14批白芍、炒白芍、酒白芍和硫熏白芍的特征图谱,标定了6个共有峰,并均被指认,分别为没食子酸、儿茶素、芍药内酯苷、芍药苷、1,2,3,4,6-五没食子酰葡萄糖和苯甲酰芍药苷,而硫熏白芍标定7个共有峰,峰7为白芍硫熏后产生;且各色谱谱峰有较好的分离,但不同炮制品特征图谱存在一定差异;含量测定结果显示,白芍炒制、酒制及硫熏后,6种成分均有不同程度的变化;借助中药色谱指纹图谱相似度评价系统和SIMCA-P13.0软件对14批白芍、炒白芍、酒白芍和硫熏白芍进行相似度和正交偏最小二乘判别(OPLS-DA)分析,所建立的白芍和炮制品及硫熏品的质量评价方法稳定性、重复性好,可用于白芍、炒白芍、酒白芍和硫熏白芍的质量控制和评价。