Expanded use of sense codons is regulated by modified cytidines in tRNA.

Codon use among the three domains of life is not confined to the universal genetic code. With only 22 tRNA genes in mammalian mitochondria, exceptions from the universal code are necessary for proper translation. A particularly interesting deviation is the decoding of the isoleucine AUA codon as methionine by the one mitochondrial-encoded tRNA(Met). This tRNA decodes AUA and AUG in both the A- and P-sites of the metazoan mitochondrial ribosome. Enrichment of posttranscriptional modifications is a commonly appropriated mechanism for modulating decoding rules, enabling some tRNA functions while restraining others. In this case, a modification of cytidine, 5-formylcytidine (f(5)C), at the wobble position-34 of human mitochondrial tRNA(f5CAU)(Met) (hmtRNA(f5CAU)(Met)) enables expanded decoding of AUA, resulting in a deviation in the genetic code. Visualization of the codon•anticodon interaction by X-ray crystallography revealed that recognition of both A and G at the third position of the codon occurs in the canonical Watson-Crick geometry. A modification-dependent shift in the tautomeric equilibrium toward the rare imino-oxo tautomer of cytidine stabilizes the f(5)C34•A base pair geometry with two hydrogen bonds.

Structural Studies of Geosmin Synthase, a Bifunctional Sesquiterpene Synthase with αα Domain Architecture That Catalyzes a Unique Cyclization-Fragmentation Reaction Sequence.

Geosmin synthase from Streptomyces coelicolor (ScGS) catalyzes an unusual, metal-dependent terpenoid cyclization and fragmentation reaction sequence. Two distinct active sites are required for catalysis: the N-terminal domain catalyzes the ionization and cyclization of farnesyl diphosphate to form germacradienol and inorganic pyrophosphate (PPi), and the C-terminal domain catalyzes the protonation, cyclization, and fragmentation of germacradienol to form geosmin and acetone through a retro-Prins reaction. A unique αα domain architecture is predicted for ScGS based on amino acid sequence: each domain contains the metal-binding motifs typical of a class I terpenoid cyclase, and each domain requires Mg(2+) for catalysis. Here, we report the X-ray crystal structure of the unliganded N-terminal domain of ScGS and the structure of its complex with three Mg(2+) ions and alendronate. These structures highlight conformational changes required for active site closure and catalysis. Although neither full-length ScGS nor constructs of the C-terminal domain could be crystallized, homology models of the C-terminal domain were constructed on the basis of ∼36% sequence identity with the N-terminal domain. Small-angle X-ray scattering experiments yield low-resolution molecular envelopes into which the N-terminal domain crystal structure and the C-terminal domain homology model were fit, suggesting possible αα domain architectures as frameworks for bifunctional catalysis.

The central role of protein S12 in organizing the structure of the decoding site of the ribosome.

The ribosome decodes mRNA by monitoring the geometry of codon-anticodon base-pairing using a set of universally conserved 16S rRNA nucleotides within the conformationally dynamic decoding site. By applying single-molecule FRET and X-ray crystallography, we have determined that conditional-lethal, streptomycin-dependence mutations in ribosomal protein S12 interfere with tRNA selection by allowing conformational distortions of the decoding site that impair GTPase activation of EF-Tu during the tRNA selection process. Distortions in the decoding site are reversed by streptomycin or by a second-site suppressor mutation in 16S rRNA. These observations encourage a refinement of the current model for decoding, wherein ribosomal protein S12 and the decoding site collaborate to optimize codon recognition and substrate discrimination during the early stages of the tRNA selection process.

Structural analysis of base substitutions in Thermus thermophilus 16S rRNA conferring streptomycin resistance.

Streptomycin is a bactericidal antibiotic that induces translational errors. It binds to the 30S ribosomal subunit, interacting with ribosomal protein S12 and with 16S rRNA through contacts with the phosphodiester backbone. To explore the structural basis for streptomycin resistance, we determined the X-ray crystal structures of 30S ribosomal subunits from six streptomycin-resistant mutants of Thermus thermophilus both in the apo form and in complex with streptomycin. Base substitutions at highly conserved residues in the central pseudoknot of 16S rRNA produce novel hydrogen-bonding and base-stacking interactions. These rearrangements in secondary structure produce only minor adjustments in the three-dimensional fold of the pseudoknot. These results illustrate how antibiotic resistance can occur as a result of small changes in binding site conformation.

Differential function of lip residues in the mechanism and biology of an anthrax hemophore.

To replicate in mammalian hosts, bacterial pathogens must acquire iron. The majority of iron is coordinated to the protoporphyrin ring of heme, which is further bound to hemoglobin. Pathogenic bacteria utilize secreted hemophores to acquire heme from heme sources such as hemoglobin. Bacillus anthracis, the causative agent of anthrax disease, secretes two hemophores, IsdX1 and IsdX2, to acquire heme from host hemoglobin and enhance bacterial replication in iron-starved environments. Both proteins contain NEAr-iron Transporter (NEAT) domains, a conserved protein module that functions in heme acquisition in Gram-positive pathogens. Here, we report the structure of IsdX1, the first of a Gram-positive hemophore, with and without bound heme. Overall, IsdX1 forms an immunoglobin-like fold that contains, similar to other NEAT proteins, a 3(10)-helix near the heme-binding site. Because the mechanistic function of this helix in NEAT proteins is not yet defined, we focused on the contribution of this region to hemophore and NEAT protein activity, both biochemically and biologically in cultured cells. Site-directed mutagenesis of amino acids in and adjacent to the helix identified residues important for heme and hemoglobin association, with some mutations affecting both properties and other mutations affecting only heme stabilization. IsdX1 with mutations that reduced the ability to associate with hemoglobin and bind heme failed to restore the growth of a hemophore-deficient strain of B. anthracis on hemoglobin as the sole iron source. These data indicate that not only is the 3(10)-helix important for NEAT protein biology, but also that the processes of hemoglobin and heme binding can be both separate as well as coupled, the latter function being necessary for maximal heme-scavenging activity. These studies enhance our understanding of NEAT domain and hemophore function and set the stage for structure-based inhibitor design to block NEAT domain interaction with upstream ligands.

Rethinking atoll futures: local resilience to global challenges.

Atoll islands are often perceived as inevitably lost due to rising sea levels. However, unlike other islands, atoll islands are dynamic landforms that have evolved, at least historically, to vertically accrete at a pace commensurate with changing sea levels. Rather than atoll islands' low elevation per se, the impairment of natural accretion processes is jeopardising their persistence. While global marine impacts are deteriorating coral reefs, local impacts also significantly affect accretion, together potentially tipping the scales toward atoll island erosion. Maintaining atoll island accretion requires intact sediment generation on coral reefs, unobstructed sediment transport from reef to island, and available vegetated deposition sites on the island. Ensuring the persistence of atoll islands must include global greenhouse gas emission reduction and local restoration of accretion processes.

GTPase activation of elongation factor EF-Tu by the ribosome during decoding.

We have used single-particle reconstruction in cryo-electron microscopy to determine a structure of the Thermus thermophilus ribosome in which the ternary complex of elongation factor Tu (EF-Tu), tRNA and guanine nucleotide has been trapped on the ribosome using the antibiotic kirromycin. This represents the state in the decoding process just after codon recognition by tRNA and the resulting GTP hydrolysis by EF-Tu, but before the release of EF-Tu from the ribosome. Progress in sample purification and image processing made it possible to reach a resolution of 6.4 A. Secondary structure elements in tRNA, EF-Tu and the ribosome, and even GDP and kirromycin, could all be visualized directly. The structure reveals a complex conformational rearrangement of the tRNA in the A/T state and the interactions with the functionally important switch regions of EF-Tu crucial to GTP hydrolysis. Thus, the structure provides insights into the molecular mechanism of signalling codon recognition from the decoding centre of the 30S subunit to the GTPase centre of EF-Tu.

Adapting federated cyberinfrastructure for shared data collection facilities in structural biology.

Early stage experimental data in structural biology is generally unmaintained and inaccessible to the public. It is increasingly believed that this data, which forms the basis for each macromolecular structure discovered by this field, must be archived and, in due course, published. Furthermore, the widespread use of shared scientific facilities such as synchrotron beamlines complicates the issue of data storage, access and movement, as does the increase of remote users. This work describes a prototype system that adapts existing federated cyberinfrastructure technology and techniques to significantly improve the operational environment for users and administrators of synchrotron data collection facilities used in structural biology. This is achieved through software from the Virtual Data Toolkit and Globus, bringing together federated users and facilities from the Stanford Synchrotron Radiation Lightsource, the Advanced Photon Source, the Open Science Grid, the SBGrid Consortium and Harvard Medical School. The performance and experience with the prototype provide a model for data management at shared scientific facilities.

Modification of 16S ribosomal RNA by the KsgA methyltransferase restructures the 30S subunit to optimize ribosome function.

All organisms incorporate post-transcriptional modifications into ribosomal RNA, influencing ribosome assembly and function in ways that are poorly understood. The most highly conserved modification is the dimethylation of two adenosines near the 3' end of the small subunit rRNA. Lack of these methylations due to deficiency in the KsgA methyltransferase stimulates translational errors during both the initiation and elongation phases of protein synthesis and confers resistance to the antibiotic kasugamycin. Here, we present the X-ray crystal structure of the Thermus thermophilus 30S ribosomal subunit lacking these dimethylations. Our data indicate that the KsgA-directed methylations facilitate structural rearrangements in order to establish a functionally optimum subunit conformation during the final stages of ribosome assembly.

Mechanism for expanding the decoding capacity of transfer RNAs by modification of uridines.

One of the most prevalent base modifications involved in decoding is uridine 5-oxyacetic acid at the wobble position of tRNA. It has been known for several decades that this modification enables a single tRNA to decode all four codons in a degenerate codon box. We have determined structures of an anticodon stem-loop of tRNA(Val) containing the modified uridine with all four valine codons in the decoding site of the 30S ribosomal subunit. An intramolecular hydrogen bond involving the modification helps to prestructure the anticodon loop. We found unusual base pairs with the three noncomplementary codon bases, including a G.U base pair in standard Watson-Crick geometry, which presumably involves an enol form for the uridine. These structures suggest how a modification in the uridine at the wobble position can expand the decoding capability of a tRNA.

A Structural Basis for Restricted Codon Recognition Mediated by 2-thiocytidine in tRNA Containing a Wobble Position Inosine.

Three of six arginine codons (CGU, CGC, and CGA) are decoded by two Escherichia coli tRNAArg isoacceptors. The anticodon stem and loop (ASL) domains of tRNAArg1 and tRNAArg2 both contain inosine and 2-methyladenosine modifications at positions 34 (I34) and 37 (m2A37). tRNAArg1 is also modified from cytidine to 2-thiocytidine at position 32 (s2C32). The s2C32 modification is known to negate wobble codon recognition of the rare CGA codon by an unknown mechanism, while still allowing decoding of CGU and CGC. Substitution of s2C32 for C32 in the Saccharomyces cerevisiae tRNAIleIAU anticodon stem and loop domain (ASL) negates wobble decoding of its synonymous A-ending codon, suggesting that this function of s2C at position 32 is a generalizable property. X-ray crystal structures of variously modified ASLArg1ICG and ASLArg2ICG constructs bound to cognate and wobble codons on the ribosome revealed the disruption of a C32-A38 cross-loop interaction but failed to fully explain the means by which s2C32 restricts I34 wobbling. Computational studies revealed that the adoption of a spatially broad inosine-adenosine base pair at the wobble position of the codon cannot be maintained simultaneously with the canonical ASL U-turn motif. C32-A38 cross-loop interactions are required for stability of the anticodon/codon interaction in the ribosomal A-site.

Active matrix metalloproteinase-2 promotes apoptosis of hepatic stellate cells via the cleavage of cellular N-cadherin.

BACKGROUND AND AIMS: Hepatic stellate cells (HSC) are known to synthesise excess matrix that characterises liver fibrosis and cirrhosis. Activated HSC express the matrix-degrading matrix metalloproteinase enzymes (MMPs) and their tissue inhibitors (TIMPs). During spontaneous recovery from experimental liver fibrosis, the expression of TIMP-1 declines and hepatic collagenolytic activity increases. This is accompanied by HSC apoptosis. In this study, we examine a potential mechanism whereby MMP activity might induce HSC apoptosis by cleaving N-cadherin at the cell surface. RESULTS: N-cadherin expression was upregulated in human HSC during activation in culture. Addition of function-blocking antibodies or a peptide targeting the extracellular domain of N-cadherin, to cultured HSC, promoted apoptosis. During apoptosis, there was cleavage of N-cadherin into 20-100 kDa fragments. MMP-2 became activated early during HSC apoptosis and directly cleaved N-cadherin in vitro. Addition of activated MMP-2 to HSCs in culture resulted in enhanced apoptosis and loss of N-cadherin. CONCLUSIONS: Together, these studies identify a role for both N-cadherin and MMP-2 in mediating HSC apoptosis, where N-cadherin works to provide a cell survival stimulus and MMP-2 promotes HSC apoptosis concomitant with N-cadherin degradation.

Structure of a purine-purine wobble base pair in the decoding center of the ribosome.

Here we report the crystal structures of I.C and I.A wobble base pairs in the context of the ribosomal decoding center, clearly showing that the I.A base pair is of an I(anti).A(anti) conformation, as predicted by Crick. Additionally, the structures enable the observation of changes in the anticodon to allow purine-purine base pairing, the 'widest' base pair geometry allowed in the wobble position.

The role of modifications in codon discrimination by tRNA(Lys)UUU.

The natural modification of specific nucleosides in many tRNAs is essential during decoding of mRNA by the ribosome. For example, tRNA(Lys)(UUU) requires the modification N6-threonylcarbamoyladenosine at position 37 (t(6)A37), adjacent and 3' to the anticodon, to bind AAA in the A site of the ribosomal 30S subunit. Moreover, it can only bind both AAA and AAG lysine codons when doubly modified with t(6)A37 and either 5-methylaminomethyluridine or 2-thiouridine at the wobble position (mnm(5)U34 or s(2)U34). Here we report crystal structures of modified tRNA anticodon stem-loops bound to the 30S ribosomal subunit with lysine codons in the A site. These structures allow the rationalization of how modifications in the anticodon loop enable decoding of both lysine codons AAA and AAG.

PHOTOSTENT-02: porfimer sodium photodynamic therapy plus stenting versus stenting alone in patients with locally advanced or metastatic biliary tract cancer.

BACKGROUND: Endobiliary stenting is standard practice for palliation of obstructive jaundice due to biliary tract cancer (BTC). Photodynamic therapy (PDT) may also improve biliary drainage and previous small studies suggested survival benefit. AIMS: To assess the difference in outcome between patients with BTC undergoing palliative stenting plus PDT versus stenting alone. METHODS: 92 patients with confirmed locally advanced or metastatic BTC, ECOG performance status 0-3 and adequate biliary drainage were randomised (46 per group) to receive porfimer sodium PDT plus stenting or stenting alone. The primary end point was overall survival (OS). Toxicity and progression-free survival (PFS) were secondary end points. Treatment arms were well balanced for baseline factors and prior therapy. RESULTS: No significant differences in grade 3-4 toxicities and no grade 3-4 adverse events due to PDT were observed. Thirteen (28%) PDT patients and 24 (52%) stent alone patients received subsequent palliative chemotherapy. After a median follow-up of 8.4 months, OS and PFS were worse in patients receiving PDT compared with stent alone group (OS median 6.2 vs 9.8 months (HR 1.56, 95% CI 1.00 to 2.43, p=0.048) and PFS median 3.4 vs 4.3 months (HR 1.43, 95% CI: 0.93 to 2.18, p=0.10), respectively). CONCLUSION: In patients with locally advanced or metastatic BTC, PDT was associated with worse outcome than stenting alone, explained only in part by the differences in chemotherapy treatments. We conclude that optimal stenting remains the treatment of choice for malignant biliary obstruction and the use of PDT for this indication cannot be recommended outside of clinical trials. TRIAL REGISTRATION NUMBER: ISRCTN 87712758; EudraCT 2005-001173-96; UKCRN ID: 1461.

The role of intercalating residues in chromosomal high-mobility-group protein DNA binding, bending and specificity.

Ubiquitous high-mobility-group (HMGB) chromosomal proteins bind DNA in a non-sequence- specific fashion to promote chromatin function and gene regulation. Minor groove DNA binding of the HMG domain induces substantial DNA bending toward the major groove, and several interfacial residues contribute by DNA intercalation. The role of the intercalating residues in DNA binding, bending and specificity was systematically examined for a series of mutant Drosophila HMGB (HMG-D) proteins. The primary intercalating residue of HMG-D, Met13, is required both for high-affinity DNA binding and normal DNA bending. Leu9 and Tyr12 directly interact with Met13 and are required for HMG domain stability in addition to linear DNA binding and bending, which is an important function for these residues. In contrast, DNA binding and bending is retained in truncations of intercalating residues Val32 and Thr33 to alanine, but DNA bending is decreased for the glycine substitutions. Furthermore, substitution of the intercalating residues with those predicted to be involved in the specificity of the HMG domain transcription factors results in increased DNA affinity and decreased DNA bending without increased specificity. These studies reveal the importance of residues that buttress intercalating residues and suggest that features of the HMG domain other than a few base-specific hydrogen bonds distinguish the sequence-specific and non-sequence-specific HMG domain functions.

Developing strategies for liver fibrosis treatment.

Liver fibrosis represents a major worldwide healthcare burden. Current therapy is limited to removing the causal agent. This approach is successful in some diseases; particularly haemochromatosis and chronic viral hepatitis. However, for many patients treatment is not possible, while other patients present to medical attention at an advanced stage of fibrosis. There is therefore a great need for novel therapies for liver fibrosis. The hepatic stellate cell has been recognised to be responsible for most of the excess extracellular matrix observed in chronic liver fibrosis. The detailed understanding of hepatic stellate cell biology has allowed the rational design of novel antifibrotic therapies. This review describes for the general reader the novel emerging therapies for liver fibrosis.

N-Cadherin cleavage during activated hepatic stellate cell apoptosis is inhibited by tissue inhibitor of metalloproteinase-1.

Apoptosis of hepatic stellate cells (HSC) has previously been shown to occur during spontaneous resolution of experimental liver fibrosis. TIMP-1 has also been shown to have a key role because of its ability to inhibit apoptosis of HSC via matrix metalloproteinase (MMP) inhibition. This has led to further study of novel substrates for MMPs that might impact on HSC survival. N-Cadherin is known to mediate cell-cell contacts in fibroblasts. In this study we demonstrate that N-Cadherin is expressed by activated rat HSC. Furthermore, during apoptosis of HSC, the N-Cadherin is cleaved into smaller fragments. Apoptosis of HSC may be inhibited by TIMP-1. This is associated with reduced fragmentation of N-Cadherin. N-Cadherin may have an important role in supporting HSC survival while N-Cadherin cleavage may play a part in promoting HSC apoptosis in recovery from liver fibrosis.

A case-control study of childhood environmental risk factors for the development of inflammatory bowel disease.

OBJECTIVE: To clarify the relationship between childhood environment and the risk of subsequent development of Crohn's disease or ulcerative colitis. DESIGN AND OUTCOME MEASURES: A case-control study, assessing the risk of inflammatory bowel disease in relation to a series of historical and serological markers of childhood circumstance, analysed using the maximum likelihood form of conditional logistic regression. SETTING: District general hospital (secondary care institution). PARTICIPANTS: Subjects with Crohn's disease (n = 139) or ulcerative colitis (n = 137) aged between 16 and 45 years, each matched for sex and age with an outpatient control. RESULTS: Helicobacter seroprevalence was substantially reduced in Crohn's disease (OR 0.18; 95% CI, 0.06-0.52) but not in ulcerative colitis (OR 0.91; 95% CI, 0.38-2.16). In ulcerative colitis, a strong negative association with childhood appendectomy was confirmed (OR 0.05; 95% CI, 0.01-0.51). Crohn's disease was associated with childhood eczema (OR 2.81; 95% CI, 1.23-6.42) and the frequent use of a swimming pool (OR 2.90; 95% CI 1.21-6.91). There was no association between hepatitis A seroprevalence and either disease. CONCLUSION: The findings are consistent with the hypothesis that improved childhood living conditions are associated with increased risk of Crohn's disease. The study confirms that the negative association between appendectomy and ulcerative colitis relates primarily to events in childhood. Overall, the findings strongly support the assertion that childhood environment is an important determinant of the risk of inflammatory bowel disease in later life, with quite distinct risk factors for ulcerative colitis and Crohn's disease.

Bidirectional imaging and modeling of skin texture.

In this paper, we present a method of skin imaging called bidirectional imaging that captures significantly more properties of appearance than standard imaging. The observed structure of the skin's surface is greatly dependent on the angle of incident illumination and the angle of observation. Specific protocols to achieve bidirectional imaging are presented and used to create the Rutgers Skin Texture Database (clinical component). This image database is the first of its kind in the dermatology community. Skin images of several disorders under multiple controlled illumination and viewing directions are provided publicly for research and educational use. Using this skin texture database, we employ computational surface modeling to perform automated skin texture classification. The classification experiments demonstrate the usefulness of the modeling and measurement methods.