4sUDRB-seq: measuring genomewide transcriptional elongation rates and initiation frequencies within cells

Background

Synthetic biology is a discipline that includes making life forms artificially from chemicals. Here, a DNA molecule was enzymatically synthesized in vitro from DNA templates made from oligonucleotides representing the text of the first Tobacco mosaic virus (TMV) sequence elucidated in 1982. No infectious DNA molecule of that seminal reference sequence exists, so the goal was to synthesize it and then build viral chimeras.

Results

RNA was transcribed from synthetic DNA and encapsidated with capsid protein in vitro to make synthetic virions. Plants inoculated with the virions did not develop symptoms. When two nucleotide mutations present in the original sequence, but not present in most other TMV sequences in GenBank, were altered to reflect the consensus, the derivative synthetic virions produced classic TMV symptoms. Chimeras were then made by exchanging TMV capsid protein DNA with Tomato mosaic virus (ToMV) and Barley stripe mosaic virus (BSMV) capsid protein DNA. Virus expressing ToMV capsid protein exhibited altered, ToMV-like symptoms in Nicotiana sylvestris. A hybrid ORF6 protein unknown to nature, created by substituting the capsid protein genes in the virus, was found to be a major symptom determinant in Nicotiana benthamiana. Virus expressing BSMV capsid protein did not have an extended host range to barley, but did produce novel symptoms in N. benthamiana.

Conclusions

This first report of the chemical synthesis and artificial assembly of a plant virus corrects a long-standing error in the TMV reference genome sequence and reveals that unnatural hybrid virus proteins can alter symptoms unexpectedly.     

Evolutionary and structural annotation of disease-associated mutations in human aminoacyl-tRNA synthetases

Background

Mutation(s) in proteins are a natural byproduct of evolution but can also cause serious diseases. Aminoacyl-tRNA synthetases (aaRSs) are indispensable components of all cellular protein translational machineries, and in humans they drive translation in both cytoplasm and mitochondria. Mutations in aaRSs have been implicated in a plethora of diseases including neurological conditions, metabolic disorders and cancer.

Results

We have developed an algorithmic approach for genome-wide analyses of sequence substitutions that combines evolutionary, structural and functional information. This pipeline enabled us to super-annotate human aaRS mutations and analyze their linkage to health disorders. Our data suggest that in some but not all cases, aaRS mutations occur in functional and structural sectors where they can manifest their pathological effects by altering enzyme activity or causing structural instability. Further, mutations appear in both solvent exposed and buried regions of aaRSs indicating that these alterations could lead to dysfunctional enzymes resulting in abnormal protein translation routines by affecting inter-molecular interactions or by disruption of non-bonded interactions. Overall, the prevalence of mutations is much higher in mitochondrial aaRSs, and the two most often mutated aaRSs are mitochondrial glutamyl-tRNA synthetase and dual localized glycyl-tRNA synthetase. Out of 63 mutations annotated in this work, only 12 (~20%) were observed in regions that could directly affect aminoacylation activity via either binding to ATP/amino-acid, tRNA or by involvement in dimerization. Mutations in structural cores or at potential biomolecular interfaces account for ~55% mutations while remaining mutations (~25%) remain structurally un-annotated.

Conclusion

This work provides a comprehensive structural framework within which most defective human aaRSs have been structurally analyzed. The methodology described here could be employed to annotate mutations in other protein families in a high-throughput manner.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1063) contains supplementary material, which is available to authorized users.     

Novel and unique domains in aminoacyl-tRNA synthetases from human fungal pathogens Aspergillus niger,Candida albicans and Cryptococcus neoformans

Background

Some species of fungi can cause serious human diseases, particularly to immuno-compromised individuals. Opportunistic fungal infections are a leading cause of mortality, and present an emerging challenge that requires development of new and effective therapeutics. Aminoacyl-tRNA synthetases (aaRSs) are indispensable components of cellular protein translation machinery and can be targeted for discovery of novel anti-fungal agents.

Results

Validation of aaRSs as potential drug targets in pathogenic microbes prompted us to investigate the genomic distribution of aaRSs within three fungi that infect humans – A. niger, C. albicans and C. neoformans. Hidden Markov Models were built for aaRSs and related proteins to search for homologues in these fungal genomes. Here, we provide a detailed and comprehensive annotation for 3 fungal genome aaRSs and their associated proteins. We delineate predicted localizations, subdomain architectures and prevalence of unusual motifs within these aaRSs. Several fungal aaRSs have unique domain appendages of unknown function e.g. A. niger AsxRS and C. neoformans TyrRS have additional domains that are absent from human homologs.

Conclusions

Detailed comparisons of fungal aaRSs with human homologs suggest key differences that could be exploited for specific drug targeting. Our cataloging and structural analyses provide a comprehensive foundation for experimentally dissecting fungal aaRSs that may enable development of new anti-fungal agents.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1069) contains supplementary material, which is available to authorized users.     

Structural and functional analysis of Vitamin K2 synthesis protein MenD

Here we describe in detail the crystal structures of the Vitamin K₂ synthesis protein MenD, from Escherichia coli, in complex with thiamine diphosphate (ThDP) and oxoglutarate, and the effects of cofactor and substrate on its structural stability. This is the first reported structure of MenD in complex with oxoglutarate. The residues Gly472 to Phe488 of the active site region are either disordered, or in an open conformation in the MenD oxoglutarate complex structure, but adopt a closed conformation in the MenD ThDP complex structure. Biospecific-interaction analysis using surface plasmon resonance (SPR) technology reveals an affinity for ThDP and oxoglutarate in the nanomolar range. Biochemical and structural analysis confirmed that MenD is highly dependent on ThDP for its structural stability. Our structural results combined with the biochemical assay reveal novel features of the enzyme that could be utilized in a program of rational structure-based drug design, as well as in helping to enhance our knowledge of the menaquinone synthesis pathway in greater detail.     

Allelopathic effects of Cyperus rotundus extract in vitro and ex vitro on banana

Allelopathic effects of Cyperus rotundus on banana ‘Grande Naine’ were studied in vitro as well as ex vitro. To study the allelopathic effects in vitro, the Cyperus extract was added to the multiplication medium during preparation before adjusting the pH. Of the four concentrations, 0.2 and 0.6% decreased the shoot multiplication and shoot length but 1 and 2% extract completely inhibited the shoot multiplication but induced rooting in 50 and 28% shoots, respectively. For the ex vitro studies, the Cyperus extract was added to the hydroponic medium during the hardening of in vitro raised banana plantlets. The extract of 1 and 2% concentrations decreased the shoot and root length, number of leaves and new roots, fresh weight, total chlorophyll and protein. The fluorescence intensity ratio was successively increased resulting in decreased photosynthesis.     

Molecular characterization of Mycobacterium tuberculosis cystathionine gamma synthase—Apo- and holoforms

Multiple probes like absorbance, circular dichroism, fluorescence and biochemical changes have been exploited to understand the role of PLP (pyridoxal 5′ phosphate) in guanidine hydrochloride (GdnHCl) mediated unfolding and refolding processes of cystathionine gamma synthase from Mycobacterium tuberculosis (MtCGS). Unfolding by GdnHCl inactivates the enzyme due to loss of ketoenamine tautomer. Though PLP induces difference in secondary structure content, it is unable to provide stabilizing effect during the overall secondary structure unfolding process. But it induces tertiary structure stability of the protein thereby counteracting the deleterious effect of denaturant. In silico modelling and cofactor docking provide insights into molecular structure of the enzyme.     

C<Subscript>1</Subscript>-/C<Subscript>2</Subscript>-aromatic-imino-glyco-conjugates: experimental and computational studies of binding,inhibition and docking aspects towards glycosidases isolated from soybean and jack bean

Several C₁-imino conjugates of d-galactose, d-lactose and d-ribose, where the nitrogen center was substituted by the salicylidene or naphthylidene, were synthesized and characterized. Similar C₂-imino conjugates of d-glucose have also been synthesized. All the glyco-imino-conjugates, which are transition state analogues, exhibited 100% inhibition of the activity towards glycosidases extracted from soybean and jack bean meal. Among these, a galactosyl-napthyl-imine-conjugate (1c) showed 50% inhibition of the activity of pure α-mannosidase from jack bean at 22 ± 2.5 μM, and a ribosyl-naphthyl-imine-conjugate (3c) showed at 31 ± 5.5 μM and hence these conjugates are potent inhibitors of glycosidases. The kinetic studies suggested non-competitive inhibition by these conjugates. The studies are also suggestive of the involvement of aromatic, imine and carbohydrate moieties of the glyco-imino-conjugates in the effective inhibition. The binding of glyco-imino-conjugate has been established by extensive studies carried out using fluorescence emission and isothermal titration calorimetry. The conformational changes resulted in the enzyme upon interaction of these derivatives has been established by studying the fluorescence quench of the enzyme by KI as well as from the secondary structural changes noticed in CD spectra. All these studies revealed the difference in the binding strengths of the naphthylidene vs. salicylidene as well as galactosyl vs. lactosyl moieties present in these conjugates. The differential inhibition of these glyco-conjugates has been addressed by quantifying the specific interactions present between the glyco-conjugates and the enzyme by using rigid docking studies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">Burkholderia cenocepacia</Emphasis>: a new biocatalyst for efficient bioreduction of ezetimibe intermediate

Ezetimibe is a selective acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitor used in hypercholesterolemia. Synthesis of ezetimibe requires enantiopure 3-[5-(4-fluorophenyl)-5(S)-hydroxypentanoyl]-4(S)-4-phenyl-1,3-oxazolidin-2-one (FOP alcohol) as a crucial intermediate which is produced by reduction of the corresponding prochiral ketone (FOP dione). A new biocatalyst from acclimatized soil was screened for bioreduction of the above prochiral ketone. The microorganism was identified by 16S mRNA sequencing, as Burkholderia cenocepacia. Various physicochemical conditions were optimized to increase cellmass and enzyme activity. The overall increase in cellmass concentration and enzyme activity was 2.06 and 1.85-fold, respectively. Various reaction conditions, for example pH, temperature, agitation, and cellmass concentration, were optimized for maximum product yield (chiral alcohol) with excellent enantioselectivity. Best reduction was achieved in phosphate buffer (50 mM, pH 8.0) at 40°C (200 rpm) and the yield of enantiopure alcohol from the corresponding prochiral ketone was 54%. This biocatalyst was also used for the reduction of various other prochiral ketones.     

Realistic threshold policy with hysteresis to control predator–prey continuous dynamics

This paper introduces a threshold policy with hysteresis (TPH) for the control of one-predator one-prey models. The models studied are the Lotka–Volterra and Rosenzweig–MacArthur two species density-dependent predator–prey models and the Arditi–Ginzburg nondimensional ratio-dependent model. The proposed policy (TPH) changes the dynamics of the system in such a way that a bounded oscillation is achieved confined to a region that does not allow extinction of either species. The policy can be designed by a suitable choice of so called virtual equilibrium points in a simple and intuitive manner.