Crystal structure of Rsr, an ortholog of the antigenic Ro protein, links conformational flexibility to RNA binding activity

Ro ribonucleoproteins are a class of antigenic ribonucleoproteins associated with rheumatic autoimmune diseases like systemic lupus erythematosus and Sj?grens syndrome in humans. Ro ribonucleoproteins are mostly composed of the 60-kDa Ro protein and small cytoplasmic RNAs, called Y RNAs, of unknown function. In eukaryotes, where Ro has been found to associate with damaged or mutant RNAs, it has been suggested that Ro may play a role in RNA quality control. In the radiation-resistant bacterium Deinococcus radiodurans and some eukaryotes, Ro has also been implicated in cell survival following UV damage. Here we present the first high resolution structure of a prokaryotic Ro ortholog, Rsr from D. radiodurans. The structure has been solved to 1.9 A resolution and shows distinct differences when compared with the eukaryotic apo- and RNA-bound Ro structures. Rsr is composed of two domains: a helical RNA binding domain and a mixed "von Willebrand factor A-like" domain containing a divalent metal binding site. Although the individual domains of Rsr are similar to the eukaryotic Ro, significantly large differences are seen at the interface of the two domains. Since this interface communicates with the conserved central cavity of Ro, which is implicated in RNA binding, changes at this interface could potentially influence RNA binding by Ro. Although the apo-Rsr protein is monomeric, Rsr binds Y RNA to form multimers of approximately 12 molecules of a 1:1 Rsr-Y RNA complex. Rsr binds D. radiodurans Y RNA with low nanomolar affinity, comparable with previously characterized eukaryotic Ro orthologs.     

A recombinant glycine receptor fragment forms homo-oligomers distinct from its GABA(A) counterpart

The ligand-gated ion channel receptor superfamily includes receptors for glycine, GABA, acetylcholine and serotonin. Whereas the acetylcholine and serotonin receptors mediate excitory neurotransmissions, both glycine and GABA(A) receptors are inhibitory. In this study, a fragment of the human glycine receptor alpha1 subunit, consisting of residues Ala165-Met291 (numbering based on the precursor protein), was hyperexpressed for the first time in Escherichia coli. This fragment is highly homologous in sequence to the corresponding fragment of the GABA(A) receptor. The recombinant fragment was found to have stable beta-rich secondary structure, similar to that found for the homologous GABA(A) receptor fragment, and ordered tertiary packing, suggesting a stable structural domain. Results from laser scattering studies suggest that the fragment forms trimers in solution. In addition, SDS-induced changes in secondary structure were found to occur prior to changes in oligomerization status, suggesting that oligomerization was secondary structure dependent. A study of quaternary structure using single particle analysis electron microscopy (EM) also suggested that the fragment formed homo-trimers. One trimer measures approximately 7.5 nm in diameter with a central cavity approximately 1.5 nm across. This is the first EM study on a single domain of the glycine receptor and the result is in contrast to the pentameric assembly of the equivalent GABA(A) receptor fragment reported by us earlier. The fact that this fragment alone could form oligomers in vitro suggests that amino acid residues within this segment may be involved in the oligomerization of the glycine receptor in vivo. Furthermore, the finding that two cousin receptor fragments form distinct quaternary structures indicates that sequence similarity does not necessarily imply quaternary structure similarity and, hence, care must be taken when applying a structure model derived from studies of individual receptors to the whole ligand-gated ion channel superfamily.     

Crystal structure of the Escherichia coli dcm very-short-patch DNA repair endonuclease bound to its reaction product-site in a DNA superhelix

Very-short-patch repair (Vsr) enzymes occur in a variety of bacteria, where they initiate nucleotide excision repair of G:T mismatches arising by deamination of 5-methyl-cytosines in specific regulatory sequences. We have now determined the structure of the archetypal dcm-Vsr endonuclease from Escherichia coli bound to the cleaved authentic hemi-deaminated/hemi-methylated dcm sequence 5′-C-OH-3′ 5′-p-T-p-A-p-G-p-G-3′/3′-G-p-G-p-T-p^Me5C-p-C formed by self-assembly of a 12mer oligonucleotide into a continuous nicked DNA superhelix. The structure reveals the presence of a Hoogsteen base pair within the deaminated recognition sequence and the substantial distortions of the DNA that accompany Vsr binding to product sites.     

Isolation and functional analysis of a Brassica juncea gene encoding a com-ponent of auxin efflux carrier

Polar auxin transport plays a divergent role in plant growth and developmental processes including root and embryo development, vascular pattern formation and cell elongation. Recently isolated Arabidopsis pin gene family was believed to encode a component of auxin efflux carrier (G(?)lweiler et al, 1998). Based on the Arabidopsis pin1 sequence we have isolated a Brassica juncea cDNA (designated Bjpin1), which encoded a 70-kDa putative auxin efflux carrier. Deduced BjPIN1 shared 65% identities at protein level with AtPINl and was highly homologous to other putative PIN proteins of Arabidopsis (with highest homology to AtPIN3). Hydrophobic analysis showed similar structures between BjPINl and AtPIN proteins. Presence of 6 exons (varying in size between 65 bp and 1229 bp) and 5 introns (sizes between 89 bp and 463 bp) in the genomic fragment was revealed by comparing the genomic and cDNA sequences. Northern blot analysis indicated that Bjpin1 was expressed in most of the tissues tested, with a relatively h     

MPP: a microarray-to-phylogeny pipeline for analysis of gene and marker content datasets

MPP is a Java application, encompassing both new and established algorithms, for the analysis of gene and marker content datasets arising from high-throughput microarray techniques. MPP analyses flat file output from microarray experiments to determine the probability of the presence or absence of genes or markers within a genome. MPP can construct gene or marker content datasets for a number of genomes and can use the data to estimate an evolutionary tree or network. Results from gene content analyses may be validated by comparing them to known gene contents. MPP was initially developed to analyse data derived from comparative genome hybridization (CGH) microarray experiments in fungi and bacteria. It has recently been adapted to analyse retrotransposon-based insertion polymorphism (RBIP) marker scores derived from tagged microarray marker (TAM) experiments in pea. New analytical procedures may be added easily to MPP as plugins in order to increase the scope of the software. AVAILABILITY: MPP source code, executables and online help are available at http://cbr.jic.ac.uk/dicks/software/     

特定人群超重及肥胖与相关疾病患病分析

目的：分析特定人群超重患病率,以及超重与高血压、糖尿病、血脂异常、脂肪肝等相关疾病的关系,为及早预防慢性非传染性疾病奠定基础。方法：对平房地区采取长效避孕措施的603名户籍农村已婚育龄妇女进行健康体检,按体重指数（BMI）分为正常组、超重组和肥胖组,比较各组间高血压、高血糖、高血脂、脂肪肝等相关疾病检出率的差异。结果：特定人群超重发病率及超重相关疾病检出率的差异均具有统计学意义（P〈0．01）。结论：平房地区特定人群超重及肥胖发病率未明显高于国内平均水平及全市水平。但超重及肥胖与高血压、糖尿病、血脂异常、脂肪肝等疾病存在较大相关关系,为了进一步降低心脑血管高危因素和死亡率。需采取早期、有效的措施控制超重和肥胖倾向。     

Molecular basis of toxicity of Clostridium perfringens epsilon toxin

Clostridium perfringens ε-toxin is produced by toxinotypes B and D strains. The toxin is the aetiological agent of dysentery in newborn lambs but is also associated with enteritis and enterotoxaemia in goats, calves and foals. It is considered to be a potential biowarfare or bioterrorism agent by the US Government Centers for Disease Control and Prevention. The relatively inactive 32.9 kDa prototoxin is converted to active mature toxin by proteolytic cleavage, either by digestive proteases of the host, such as trypsin and chymotrypsin, or by C. perfringens λ-protease. In vivo, the toxin appears to target the brain and kidneys, but relatively few cell lines are susceptible to the toxin, and most work has been carried out using Madin-Darby canine kidney (MDCK) cells. The binding of ε-toxin to MDCK cells and rat synaptosomal membranes is associated with the formation of a stable, high molecular weight complex. The crystal structure of ε-toxin reveals similarity to aerolysin from Aeromonas hydrophila, parasporin-2 from Bacillus thuringiensis and a lectin from Laetiporus sulphureus. Like these toxins, ε-toxin appears to form heptameric pores in target cell membranes. The exquisite specificity of the toxin for specific cell types suggests that it binds to a receptor found only on these cells.     

Asymmetric binding of membrane proteins to GroEL

The interaction of GroEL with non-native soluble proteins has been studied intensively and structure-function relationships have been established in considerable detail. Recently, we found that GroEL is also able to bind membrane proteins in the absence of detergents and deliver them to liposomes in a biologically active state. Here, we report that three well-studied membrane proteins (bacteriorhodopsin, LacY, and the bacteriophage lambda holin) bind asymmetrically to tetradecameric GroEL. Each of the membrane proteins was visualized in one of the center cavities of GroEL using single particle analysis.     

Insights into the structure of human cytomegalovirus large terminase subunit pUL56

Terminases are a class of proteins which catalyze the generation of unit-length genomes during DNA packaging. These essential proteins are conserved throughout the herpesviruses and many double-stranded DNA bacteriophages. We have determined the structure of the large terminase subunit pUL56 of human cytomegalovirus, a highly pathogenic virus, to 2.6 nm resolution. Image analysis of purified pUL56 suggests that the molecule exists as a dimer formed by the association of two ring-like structures positioned on top of each other and connected by a pronounced density on one side. The 3D reconstruction of pUL56 provides first structural insights into the active protein.     

A comparative study of uracil-DNA glycosylases from human and herpes simplex virus type 1

Uracil-DNA glycosylase (UNG) is the key enzyme responsible for initiation of base excision repair. We have used both kinetic and binding assays for comparative analysis of UNG enzymes from humans and herpes simplex virus type 1 (HSV-1). Steady-state fluorescence assays showed that hUNG has a much higher specificity constant (k(cat)/K(m)) compared with the viral enzyme due to a lower K(m). The binding of UNG to DNA was also studied using a catalytically inactive mutant of UNG and non-cleavable substrate analogs (2'-deoxypseudouridine and 2'-alpha-fluoro-2'-deoxyuridine). Equilibrium DNA binding revealed that both human and HSV-1 UNG enzymes bind to abasic DNA and both substrate analogs more weakly than to uracil-containing DNA. Structure determination of HSV-1 D88N/H210N UNG in complex with uracil revealed detailed information on substrate binding. Together, these results suggest that a significant proportion of the binding energy is provided by specific interactions with the target uracil. The kinetic parameters for human UNG indicate that it is likely to have activity against both U.A and U.G mismatches in vivo. Weak binding to abasic DNA also suggests that UNG activity is unlikely to be coupled to the subsequent common steps of base excision repair.