Sequence-independent Fashion Research Articles

Photochemical Action Plots Map Orthogonal Reactivity in Photochemical Release Systems.

The wavelength-by-wavelength resolved photoreactivity of two photo-caged carboxylic acids, i. e. 7-(diethylamino)-coumarin- and 3-perylene-modified substrates, is investigated via photochemical action plots. The observed wavelength-dependent reactivity of the chromophores is contrasted with their absorption profile. The photochemical action plots reveal a remarkable mismatch between the maximum reactivity and the absorbance. Through the action plot data, the study is able to uncover photochemical reactivity maxima at longer and shorter wavelengths, where the molar absorptivity of the chromophores is strongly reduced. Finally, the laser experiments are translated to light emitting diode (LED)irradiation and show efficient visible-light-induced release in a near fully wavelength-orthogonal, sequence-independent fashion (λLED1=405nm, λLED2=505nm) with both chromophores in the same reaction solution. The herein pioneered wavelength orthogonal release systems open an avenue for releasing two different molecular cargos with visible light in a fully orthogonal fashion.

In vitro isolation of small-molecule-binding aptamers with intrinsic dye-displacement functionality.

Aptamer-based sensors offer a powerful tool for molecular detection, but the practical implementation of these biosensors is hindered by costly and laborious sequence engineering and chemical modification procedures. We report a simple strategy for directly isolating signal-reporting aptamers in vitro through systematic evolution of ligands by exponential enrichment (SELEX) that transduce binding events into a detectable change of absorbance via target-induced displacement of a small-molecule dye. We first demonstrate that diethylthiatricarbocyanine (Cy7) can stack into DNA three-way junctions (TWJs) in a sequence-independent fashion, greatly altering the dye's absorbance spectrum. We then design a TWJ-containing structured library and isolate an aptamer against 3,4-methylenedioxypyrovalerone (MDPV), a synthetic cathinone that is an emerging drug of abuse. This aptamer intrinsically binds Cy7 within its TWJ domain, but MDPV efficiently displaces the dye, resulting in a change in absorbance within seconds. This assay is label-free, and detects nanomolar concentrations of MDPV. It also recognizes other synthetic cathinones, offering the potential to detect newly-emerging designer drugs, but does not detect structurally-similar non-cathinone compounds or common cutting agents. Moreover, we demonstrate that the Cy7-displacement colorimetric assay is more sensitive than a conventional strand-displacement fluorescence assay. We believe our strategy offers an effective generalized approach for the development of sensitive dye-displacement colorimetric assays for other small-molecule targets.

Structural insights into RNA encapsidation and helical assembly of the Toscana virus nucleoprotein

Toscana virus is an emerging bunyavirus in Mediterranean Europe where it accounts for 80% of pediatric meningitis cases during the summer. The negative-strand ribonucleic acid (RNA) genome of the virus is wrapped around the virally encoded nucleoprotein N to form the ribonucleoprotein complex (RNP). We determined crystal structures of hexameric N alone (apo) and in complex with a nonameric single-stranded RNA. RNA is sequestered in a sequence-independent fashion in a deep groove inside the hexamer. At the junction between two adjacent copies of Ns, RNA binding induced an inter-subunit rotation, which opened the RNA-binding tunnel and created a new assembly interface at the outside of the hexamer. Based on these findings, we suggest a structural model for how binding of RNA to N promotes the formation of helical RNPs, which are a characteristic hallmark of many negative-strand RNA viruses.

A Solid-Phase Platform for Combinatorial and Scarless Multipart Gene Assembly

With the emergence of standardized genetic modules as part of the synthetic biology toolbox, the need for universal and automatable assembly protocols increases. Although several methods and standards have been developed, these all suffer from drawbacks such as the introduction of scar sequences during ligation or the need for specific flanking sequences or a priori knowledge of the final sequence to be obtained. We have developed a method for scarless ligation of multipart gene segments in a truly sequence-independent fashion. The big advantage of this approach is that it is combinatorial, allowing the generation of all combinations of several variants of two or more modules to be ligated in less than a day. This method is based on the ligation of single-stranded or double-stranded oligodeoxynucleotides (ODN) and PCR products immobilized on a solid support. Different settings were tested to optimize the solid-support ligation. Finally, to show proof of concept for this novel multipart gene assembly platform a small library of all possible combinations of 4 BioBrick modules was generated and tested.

Detection of Leishmania RNA Virus in Leishmania Parasites

BackgroundPatients suffering from cutaneous leishmaniasis (CL) caused by New World Leishmania (Viannia) species are at high risk of developing mucosal (ML) or disseminated cutaneous leishmaniasis (DCL). After the formation of a primary skin lesion at the site of the bite by a Leishmania-infected sand fly, the infection can disseminate to form secondary lesions. This metastatic phenotype causes significant morbidity and is often associated with a hyper-inflammatory immune response leading to the destruction of nasopharyngeal tissues in ML, and appearance of nodules or numerous ulcerated skin lesions in DCL. Recently, we connected this aggressive phenotype to the presence of Leishmania RNA virus (LRV) in strains of L. guyanensis, showing that LRV is responsible for elevated parasitaemia, destructive hyper-inflammation and an overall exacerbation of the disease. Further studies of this relationship and the distribution of LRVs in other Leishmania strains and species would benefit from improved methods of viral detection and quantitation, especially ones not dependent on prior knowledge of the viral sequence as LRVs show significant evolutionary divergence.Methodology/Principal FindingsThis study reports various techniques, among which, the use of an anti-dsRNA monoclonal antibody (J2) stands out for its specific and quantitative recognition of dsRNA in a sequence-independent fashion. Applications of J2 include immunofluorescence, ELISA and dot blot: techniques complementing an arsenal of other detection tools, such as nucleic acid purification and quantitative real-time-PCR. We evaluate each method as well as demonstrate a successful LRV detection by the J2 antibody in several parasite strains, a freshly isolated patient sample and lesion biopsies of infected mice.Conclusions/SignificanceWe propose that refinements of these methods could be transferred to the field for use as a diagnostic tool in detecting the presence of LRV, and potentially assessing the LRV-related risk of complications in cutaneous leishmaniasis.

Mapping the Location of Autoinhibitory B-Domain Motifs within Factor V: New Insights Into How Factor V Activation Unfolds

Abstract 495Blood coagulation factor V (FV) circulates as a procofactor with little or no procoagulant activity. Proteolytic removal of a large central B-domain converts FV (domain organization of A1-A2-B-A3-C1-C2) to an active cofactor for membrane-bound FXa to form prothrombinase. Recently we found that discrete, evolutionarily conserved sequences within the B-domain serve an autoinhibitory function and are necessary to maintain FV as an inactive procofactor. The autoinhibitory region within the B-domain (termed the procofactor regulatory region or PRR) is sequence specific and remarkably short (∼100 amino acids out of 836 from B-domain) consisting of basic (963–1008; basic region or BR) and acidic (1493–1537; acidic region or AR) sequences. Dismantling this region appears to be the driving force to unveil a high affinity binding site(s) for FXa and thus underpins the procofactor to cofactor transition. While the BR and AR seem to work together to provide “on-site” repression of cofactor activity, there is no structural information about the FV B-domain that could provide clues into how this critical region functions. Here we use B-domain fragments tethered to an artificial protease in order to map the position of the PRR relative to the rest of the FV molecule. A BR peptide with an introduced free Cys at position 990 was stoichiometrically modified with FeBABE (Fe-p-bromoacetamidobenzyl-EDTA). FeBABE is a protein cutting reagent with a sulfhydryl-reactive moiety for attachment to Cys and an EDTA-chelated iron atom which, when triggered with ascorbic acid and peroxide, generates hydroxyl radicals that cut peptide bonds in a sequence-independent fashion. When a protein labeled with FeBABE binds its target, the Fe-BABE moiety facilitates cutting if it is orientated correctly and near (<12 Å) the contact site of the target protein. In our experimental system, we employed a constitutively active B-domainless form of FV (FV-810) that harbors the AR but lacks the BR. Initial studies revealed that the BR peptide, without or tethered with FeBABE, binds to FV-810 with high affinity (nM) and inhibits cofactor activity. Proteolysis of FV-810 by BR-FeBABE, was visualized by Western blot, using monoclonal antibodies recognizing either the heavy or light chains. Using this approach, we consistently found that BR-FeBABE cut FV-810 predominantly at two sites. The first is at the C-terminal end of the B-domain. This region is enriched in acidic residues and represents the AR defined above. The second site of cleavage is within the heavy chain. While we cannot exclude the possibility that the site of contact is at the N-terminus of the A1 domain, based on the proposed 3D structure of FVa, it is more likely that the BR is binding near the C-terminal end of the A2 domain which is also enriched in acidic amino acids. Compared to a panel of FVa derivatives with variably truncated heavy chains, we suggest that BR-FeBABE cuts FV-810 near residues 675–685. Overall the data suggest that the B-domain must be folded in such a way as to place the BR in close spatial proximity to the AR at the C-terminal end of the B-domain and also to the C-terminal heavy chain region. Consistent with the idea that these interacting motifs work to keep FV as a procofactor by suppressing FXa binding, adding saturating amounts of FXa, but not the zymogen FX, prevented the BR-FeBABE induced cleavage of FV-810. Similarly, activated protein C (APC) which is thought to share a related binding site with FXa, also prevented BR-FeBABE from cutting FV-810. Addition of excess unlabeled BR peptide substantially reduced cutting of FV-810 by BR-FeBABE, indicating that proteolysis is not due to non-specific cleavage. Additionally, removing the basic charge of BR-FeBABE via acetylation eliminated binding to FV-810 and also any signs of specific cutting. Further, FV-derivatives harboring both the AR and BR in cis and hence not binding the BR peptide, were not cut by excess BR-FeBABE added in trans. In conclusion, these findings provide new insights into the spatial relationship between key regulatory B-domain motifs and the core heavy/light chain region. These interacting autoinhibitory motifs directly or indirectly obscure FXa binding and hence stabilize the inactive procofactor state of FV. These studies also document a remarkably powerful way to map the spatial relationship between regions of proteins and provide new information on coagulation factor binding sites. Disclosures:Camire:Pfizer: Patents & Royalties, Research Funding.

Linking epigenetics to lipid metabolism: Focus on histone deacetylases

A number of recent studies revealed that epigenetic modifications play a central role in the regulation of lipid and of other metabolic pathways such as cholesterol homeostasis, bile acid synthesis, glucose and energy metabolism. Epigenetics refers to aspects of genome functions regulated in a DNA sequence-independent fashion. Chromatin structure is controlled by epigenetic mechanisms through DNA methylation and histone modifications. The main modifications are histone acetylation and deacetylation on specific lysine residues operated by two different classes of enzymes: Histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively. The interaction between these enzymes and histones can activate or repress gene transcription: Histone acetylation opens and activates chromatin, while deacetylation of histones and DNA methylation compact chromatin making it transcriptionally silent. The new evidences on the importance of HDACs in the regulation of lipid and other metabolic pathways will open new perspectives in the comprehension of the pathophysiology of metabolic disorders.

Structural basis for multifunctional roles of mammalian aminopeptidase N

Mammalian aminopeptidase N (APN) plays multifunctional roles in many physiological processes, including peptide metabolism, cell motility and adhesion, and coronavirus entry. Here we determined crystal structures of porcine APN at 1.85 Å resolution and its complexes with a peptide substrate and a variety of inhibitors. APN is a cell surface-anchored and seahorse-shaped zinc-aminopeptidase that forms head-to-head dimers. Captured in a catalytically active state, these structures of APN illustrate a detailed catalytic mechanism for its aminopeptidase activity. The active site and peptide-binding channel of APN reside in cavities with wide openings, allowing easy access to peptides. The cavities can potentially open up further to bind the exposed N terminus of proteins. The active site anchors the N-terminal neutral residue of peptides/proteins, and the peptide-binding channel binds the remainder of the peptides/proteins in a sequence-independent fashion. APN also provides an exposed outer surface for coronavirus binding, without its physiological functions being affected. These structural features enable APN to function ubiquitously in peptide metabolism, interact with other proteins to mediate cell motility and adhesion, and serve as a coronavirus receptor. This study elucidates multifunctional roles of APN and can guide therapeutic efforts to treat APN-related diseases.

TbENF is an essential TbTFIIB-interacting trypanosomatid-specific factor

Trypanosoma brucei, the causative agent of African Sleeping Sickness, is replete with unique biochemistry, including unusual features of gene transcription. The parasite also contains over 4500 non-annotated genes, representing novel biochemistry yet to be explored. Using tandem affinity purification (TAP)-tagged TbTFIIB, we identified and subsequently confirmed, one of the non-annotated T. brucei proteins, Tb11.02.4300, as a TbTFIIB-interacting protein. The 49kDa protein is nuclear and essential for parasite variability as determined by RNA interference studies; hence, the nomenclature T. brucei Essential Nuclear Factor (TbENF). TbENF is shown to interact with DNA in a sequence-independent fashion under the conditions examined. Furthermore, TbENF bears motifs associated with many eukaryotic transcription factors, such as a glutamine-rich region and a leucine zipper, yet TbENF is specific to trypanosomatids making it a potentially attractive therapeutic target. Taken together, our results suggest a role for TbENF in trypanosome gene transcription.

Modular and versatile hybrid coordination motifs on alpha-helical protein surfaces.

We report here the construction of phenanthroline (Phen) and terpyridine (Terpy)-based hybrid coordination motifs (HCMs), which were installed on the surface of the four-helical bundle hemeprotein cytochrome cb(562). The resulting constructs, termed HPhen1, HPhen2, HPhen3, and HTerpy1, feature HCMs that are composed of a histidine ligand and a Phen or Terpy functionality located two helix turns away, yielding stable tri- or tetradentate coordination platforms. Our characterization of the tridentate HCMs indicates that they accommodate many divalent metal ions (Co(2+), Ni(2+), Cu(2+), Zn(2+)) with nanomolar to femtomolar affinities, lead to significant stabilization of the alpha-helical protein scaffold through metal-mediated cross-linking, assert tight control over protein dimerization, and provide stable and high-affinity binding sites for substitution-inert metal probes. Our analyses suggest that such tridentate HCMs may be used modularly on any alpha-helical protein surface in a sequence-independent fashion.

K65R and K65A Substitutions in HIV-1 Reverse Transcriptase Enhance Polymerase Fidelity by Decreasing Both dNTP Misinsertion and Mispaired Primer Extension Efficiencies

Lys65 residue, in the fingers domain of human immunodeficiency virus reverse transcriptase (RT), interacts with incoming dNTP in a sequence-independent fashion. We showed previously that a 5-amino-acid deletion spanning Lys65 and a K65A substitution both enhanced the fidelity of dNTP insertion. We hypothesized that the Lys65 residue enhances dNTP misinsertion via interactions with the γ-phosphate of the incoming dNTP. We now examine this hypothesis in pre-steady-state kinetic studies using wild-type human immunodeficiency virus-1 RT and two substitution mutants, K65A and K65R. K65R mutation did not greatly increase misinsertion fidelity, but K65A mutation led to higher incorporation fidelity. For a misinsertion to become a permanent error, it needs to be accompanied by the extension of the mispaired terminus thus formed. Both mutants and the wild-type enzyme discriminated against the mismatched primer at the catalytic step ( k pol). Additionally, K65A and K65R mutants displayed a further decrease in mismatch extension efficiency, primarily at the level of dNTP binding. We employed hydroxyl radical footprinting to determine the position of the RT on the primer/template. The wild-type and Lys65-substituted enzymes occupied the same position at the primer terminus; the presence of a mismatched primer terminus caused all three enzymes to be displaced to a − 2 position relative to the primer 3′ end. In the context of an efficiently extended mismatched terminus, the presence of the next complementary nucleotide overcame the displacement, resulting in a complex resembling the matched terminus. The results are consistent with the observed reduction in k pol in mispaired primer extension being due to the position of the enzyme at a mismatched terminus. Our work shows the influence of the stabilizing interactions of Lys65 with the incoming dNTP on two different aspects of polymerase fidelity.

SUMO-1 possesses DNA binding activity.

BackgroundConjugation of small ubiquitin-related modifiers (SUMOs) is a frequent post-translational modification of proteins. SUMOs can also temporally associate with protein-targets via SUMO binding motifs (SBMs). Protein sumoylation has been identified as an important regulatory mechanism especially in the regulation of transcription and the maintenance of genome stability. The precise molecular mechanisms by which SUMO conjugation and association act are, however, not understood.FindingsUsing NMR spectroscopy and protein-DNA cross-linking experiments, we demonstrate here that SUMO-1 can specifically interact with dsDNA in a sequence-independent fashion. We also show that SUMO-1 binding to DNA can compete with other protein-DNA interactions at the example of the regulatory domain of Thymine-DNA Glycosylase and, based on these competition studies, estimate the DNA binding constant of SUMO1 in the range 1 mM.ConclusionThis finding provides an important insight into how SUMO-1 might exert its activity. SUMO-1 might play a general role in destabilizing DNA bound protein complexes thereby operating in a bottle-opener way of fashion, explaining its pivotal role in regulating the activity of many central transcription and DNA repair complexes.

Activation and Regulation of Toll-like Receptor 9: CpGs and Beyond

Toll-like receptor 9 has been the focus of considerable research attention for the ability to modulate its activity, and subsequent innate immune responses, through DNA-based immunotherapeutics. Nucleic acids are attractive as therapeutics for their low cost, chemical stability and ease of production. While the ability for TLR9 to be differentially regulated by nucleic acids of varying sequences and structures offers flexibility for immunotherapeutic design, it also necessitates a more comprehensive characterization of these agonists in terms of how these structural parameters correlate with the activation of unique cellular responses. Despite the utilization of TLR9 agonists in human trials these issues have not been adequately addressed. While a wealth of cell stimulation experiments demonstrate the preferential ability for nucleic acids which contain unmethylated cytosine-phosphate-guanine (CpG) motifs to initiate innate immune responses this has not been supported by binding investigations from which largely contradictory information has emerged with respect to the ability of TLR9 to bind nucleic acids in a sequence-dependent fashion. Recent models help to reconcile this apparent contradiction by suggesting that while TLR9 activation is specific for CpG-containing nucleic acids, the receptor binds, and is functionally influenced by, nucleic acids in a sequence-independent fashion. We have proposed a model in which the absolute concentration of nucleic acids modulates the sensitivity of the receptor in a sequence-dependent fashion while activation is specifically achieved by CpG-containing ligands. In this review we reconsider the literature from the perspective of this new appreciation of the functional complexity of TLR9 ligand binding and higher-order regulation with discussion of the implications for immunotherapeutic targeting of TLR9.

Nucleic Acids Exert a Sequence-independent Cooperative Effect on Sequence-dependent Activation of Toll-like Receptor 9

Toll-like receptor 9 (TLR9) activates the innate immune system in response to microbial DNA or mimicking oligodeoxynucleotides. Although cell stimulation experiments demonstrate the preferential activation of TLR9 by CpG-containing nucleic acids, direct binding investigations have reached contradictory conclusions with respect to the ability of this receptor to bind nucleic acids in a sequence-specific manner. To address this apparent discrepancy, we report the purification of the soluble ectodomain of human TLR9 with characterization of its ligand binding properties. We observe that TLR9 has a high degree of specificity in its ability to bind nucleic acids that contain CpG dinucleotides as well as higher order motifs that mediate species-specific activation. However, TLR9 is also functionally influenced by nucleic acids in a sequence-independent fashion as both stimulatory and nonstimulatory nucleic acids sensitize TLR9 for in vitro ligand binding as well as in vivo activation. We propose a model in which receptor activation is achieved in a sequence-dependent manner, and sensitivity is modulated by the absolute concentration of nucleic acids in a sequence-independent fashion. This model bears resemblance to that recently proposed for Toll in that activation is a two-step process in which formation of a ligand-bound monomer precedes formation of the activated dimer. In each model receptor sensitivity is determined within the second step with the crucial distinction that Toll undergoes negative cooperativity, whereas TLR9 is sensitized through a positive cooperative effect.

Effect Of ISS And TGF-β1 On MLg Lung Fibroblast Proliferation In Vitro

RATIONALE: Since CpG-rich immunostimulatory DNA sequences (ISS) reduced peribronchial fibrosis in the chronic ovalbumin murine asthma model of airway remodeling, we hypothesized that ISS might act in part through a TLR9-dependent effect on fibroblast proliferation.METHODS: RT-PCR was used to determine whether TLR-9 was expressed by MLg, a murine lung fibroblast cell line. MLg fibroblast proliferation was assayed with a BrdU ELISA at 48h. MLg fibroblasts were incubated after a growth arrest phase with ISS 10 μg/ml (a ligand for TLR9), or M-ODN 10 μg/ml (modified oligodeoxynucleotides, which are not TLR9 ligands) with or without TGF-β1 15 ng/ml.RESULTS: MLg fibroblasts expressed TLR-9 as assessed by RT-PCR. MLg fibroblasts incubated with ISS alone had increased proliferation relative to both M-ODN (p<0.01) and untreated media (p<0.02). In contrast, MLg fibroblasts incubated with the combination of ISS and TGF-β1 had reduced proliferation relative to media (p<0.03). The combination of M-ODN and TGF-β1 also inhibited MLg fibroblast proliferation (p<0.03). TGF-β1 alone did not significantly affect proliferation of MLg fibroblasts.CONCLUSIONS: MLg fibroblasts express TLR-9 as assessed by RT-PCR. In the absence of TGF-β1, ISS induced proliferation of MLg fibroblasts in a TLR-9 dependent manner. In the presence of TGF-β1, exogenous DNA (either ISS or M-ODN) inhibited proliferation of MLg fibroblasts in a DNA sequence-independent fashion. Thus in MLg fibroblasts, ISS may either induce proliferation, or inhibit proliferation dependent upon the presence of extracellular TGF-β1. RATIONALE: Since CpG-rich immunostimulatory DNA sequences (ISS) reduced peribronchial fibrosis in the chronic ovalbumin murine asthma model of airway remodeling, we hypothesized that ISS might act in part through a TLR9-dependent effect on fibroblast proliferation. METHODS: RT-PCR was used to determine whether TLR-9 was expressed by MLg, a murine lung fibroblast cell line. MLg fibroblast proliferation was assayed with a BrdU ELISA at 48h. MLg fibroblasts were incubated after a growth arrest phase with ISS 10 μg/ml (a ligand for TLR9), or M-ODN 10 μg/ml (modified oligodeoxynucleotides, which are not TLR9 ligands) with or without TGF-β1 15 ng/ml. RESULTS: MLg fibroblasts expressed TLR-9 as assessed by RT-PCR. MLg fibroblasts incubated with ISS alone had increased proliferation relative to both M-ODN (p<0.01) and untreated media (p<0.02). In contrast, MLg fibroblasts incubated with the combination of ISS and TGF-β1 had reduced proliferation relative to media (p<0.03). The combination of M-ODN and TGF-β1 also inhibited MLg fibroblast proliferation (p<0.03). TGF-β1 alone did not significantly affect proliferation of MLg fibroblasts. CONCLUSIONS: MLg fibroblasts express TLR-9 as assessed by RT-PCR. In the absence of TGF-β1, ISS induced proliferation of MLg fibroblasts in a TLR-9 dependent manner. In the presence of TGF-β1, exogenous DNA (either ISS or M-ODN) inhibited proliferation of MLg fibroblasts in a DNA sequence-independent fashion. Thus in MLg fibroblasts, ISS may either induce proliferation, or inhibit proliferation dependent upon the presence of extracellular TGF-β1.

PF0610, a Novel Winged Helix−Turn−Helix Variant Possessing a Rubredoxin-like Zn Ribbon Motif from the Hyperthermophilic Archaeon, Pyrococcus furiosus,

PF0610, a protein from the hyperthermophile Pyrococcus furiosus, has homologues only in other archaeal species and in three species of Fe(III)-reducing bacteria. It is thought to have a helix-turn-helix (HTH) domain at the N-terminus and possesses two CXXC motifs characteristic of metal binding proteins. We have determined the solution structure of the Zn-bound protein using NMR. PF0610 is a novel winged helix-turn-helix (wHTH) protein with a rubredoxin-like Zn ribbon as its W1 segment. In addition, it possesses a large number of basic residues on its surface. Clusters of basic residues can be found on both helix H3 and the metal-binding loops of W1, suggesting that it might be a DNA-binding protein. Accordingly, gel shift assays using both linear and circular DNA showed that PF0610 does bind DNA, at least in a sequence-independent fashion. Modeling the PF0610-DNA interaction based on other wHTH protein-DNA structures revealed that besides helix H3, basic residues around the second CXXC motif in the metal-binding loop could make extensive contacts with DNA. However, the bulkiness of the W1 region implies that the DNA conformation may be distorted upon PF0610 binding. PF0610 is the first protein known to have a Zn ribbon-embedded wHTH fold and, as such, has potential roles both as a metal-dependent transcription regulator and as a component of the chromosome packing system in P. furiosus. The discovery of this novel structure represents the addition of another branch to the winged HTH protein family and could contribute to our understanding of transcription regulatory processes in P. furiosus.

Analysis of the Virus Population Present in Equine Faeces Indicates the Presence of Hundreds of Uncharacterized Virus Genomes

Virus DNA was isolated from horse faeces and cloned in a sequence-independent fashion. 268 clones were sequenced and 178140 nucleotides of sequence obtained. Statistical analysis suggests the library contains 17560 distinct clones derived from up to 233 different virus genomes. TBLASTX analysis showed that 32% of the clones had significant identity to GenBank entries. Of these 63% were viral; 20% bacterial; 7% archaeal; 6% eukarya; and 5% were related to mobile genetic elements. Fifty-two percent of the virus identities were with Siphoviridae; 26% unclassified phages; 17% Myoviridae; 4% Podoviridae; and one clone (2%) was a vertebrate Orthopoxvirus. Genes coding for predicted virus structural proteins, proteases, glycosidases and nucleic acid-binding proteins were common.

Sequence-specific High Mobility Group Box Factors Recognize 10–12-Base Pair Minor Groove Motifs

Sequence-specific High Mobility Group Box Factors Recognize 10–12-Base Pair Minor Groove Motifs

Studies of RNA Cleavage by Photolysis of N-Hydroxypyridine-2(1H)-thione. A New Photochemical Footprinting Method

N-Hydroxypyridine-2(1H)-thione (N-HPT) has been studied as a photochemical source of hydroxyl radicals for use in photoinitiated nucleic acid footprinting experiments. Steady-state photolysis of dilute aqueous solutions of N-HPT at 350 nm in the presence of a 385 nucleotide (32)P-labeled RNA, the Tetrahymena L-21 ribozyme, resulted in cleavage of the RNA at nucleotide resolution. No cleavage of the RNA occurred in the absence of light or in the absence of N-HPT. Photolysis of the analogous pyridine lacking the N-hydroxyl group did not result in detectable amounts of RNA cleavage. The addition of RNA to preirradiated solutions of N-HPT gave no apparent RNA cleavage products, suggesting that the photoproducts of N-HPT do not result in RNA modification. Cleavage of RNA, upon photolysis in the presence of N-HPT, occurred in a sequence-independent fashion with double-stranded RNA being cleaved as efficiently as single-stranded RNA. Based on these observations, we conclude that photochemically generated diffusable hydroxyl radicals are responsible for the RNA cleavage. Experiments involving the photolysis of N-HPT in the presence of the Tetrahymena ribozyme and magnesium showed a magnesium-dependent protection from RNA cleavage due to formation of a folded RNA tertiary structure. The locations and amount of protection were identical to those observed in footprinting experiments performed with other hydroxyl radical sources. The presence of N-HPT had no effect on either the rate of folding or the catalytic activity of the folded RNA, indicating that this reagent does not disrupt RNA tertiary structure or otherwise affect activity. Thus, N-HPT is established as a new reagent for use in photoinitiated RNA footprinting experiments.

SIR repression of a yeast heat shock gene: UAS and TATA footprints persist within heterochromatin.

Previous work has suggested that products of the Saccharomyces cerevisiae Silent Information Regulator (SIR) genes form a complex with histones, nucleated by cis-acting silencers or telomeres, which represses transcription in a position-dependent but sequence-independent fashion. While it is generally thought that this Sir complex works through the establishment of heterochromatin, it is unclear how this structure blocks transcription while remaining fully permissive to other genetic processes such as recombination or integration. Here we examine the molecular determinants underlying the silencing of HSP82, a transcriptionally potent, stress-inducible gene. We find that HSP82 is efficiently silenced in a SIR-dependent fashion, but only when HMRE mating-type silencers are configured both 5' and 3' of the gene. Accompanying dominant repression are novel wrapped chromatin structures within both core and upstream promoter regions. Strikingly, DNase I footprints mapping to the binding sites for heat shock factor (HSF) and TATA-binding protein (TBP) are strengthened and broadened, while groove-specific interactions, as detected by dimethyl sulfate, are diminished. Our data are consistent with a model for SIR repression whereby transcriptional activators gain access to their cognate sites but are rendered unproductive by a co-existing heterochromatic complex.