| 1. |
- Albers, Suki, et al.
(författare)
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Repurposing tRNAs for nonsense suppression
- 2021
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- Three stop codons (UAA, UAG and UGA) terminate protein synthesis and are almost exclusively recognized by release factors. Here, we design de novo transfer RNAs (tRNAs) that efficiently decode UGA stop codons in Escherichia coli. The tRNA designs harness various functionally conserved aspects of sense-codon decoding tRNAs. Optimization within the T Psi C-stem to stabilize binding to the elongation factor, displays the most potent effect in enhancing suppression activity. We determine the structure of the ribosome in a complex with the designed tRNA bound to a UGA stop codon in the A site at 2.9 angstrom resolution. In the context of the suppressor tRNA, the conformation of the UGA codon resembles that of a sense-codon rather than when canonical translation termination release factors are bound, suggesting conformational flexibility of the stop codons dependent on the nature of the A-site ligand. The systematic analysis, combined with structural insights, provides a rationale for targeted repurposing of tRNAs to correct devastating nonsense mutations that introduce a premature stop codon. Here, the authors report de novo design, optimization and characterization of tRNAs that decode UGA stop codons in E. coli. The structure of the ribosome in a complex with the designed tRNA bound to a UGA stop codon suggests that distinct A-site ligands (tRNAs versus release factors) induce distinct conformation of the stop codon within the mRNA in the decoding center.
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| 2. |
- Calabrese, Claudia, et al.
(författare)
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Genomic basis for RNA alterations in cancer
- 2020
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 578:7793, s. 129-136
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Tidskriftsartikel (refereegranskat)abstract
- Transcript alterations often result from somatic changes in cancer genomes1. Various forms of RNA alterations have been described in cancer, including overexpression2, altered splicing3 and gene fusions4; however, it is difficult to attribute these to underlying genomic changes owing to heterogeneity among patients and tumour types, and the relatively small cohorts of patients for whom samples have been analysed by both transcriptome and whole-genome sequencing. Here we present, to our knowledge, the most comprehensive catalogue of cancer-associated gene alterations to date, obtained by characterizing tumour transcriptomes from 1,188 donors of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA)5. Using matched whole-genome sequencing data, we associated several categories of RNA alterations with germline and somatic DNA alterations, and identified probable genetic mechanisms. Somatic copy-number alterations were the major drivers of variations in total gene and allele-specific expression. We identified 649 associations of somatic single-nucleotide variants with gene expression in cis, of which 68.4% involved associations with flanking non-coding regions of the gene. We found 1,900 splicing alterations associated with somatic mutations, including the formation of exons within introns in proximity to Alu elements. In addition, 82% of gene fusions were associated with structural variants, including 75 of a new class, termed 'bridged' fusions, in which a third genomic location bridges two genes. We observed transcriptomic alteration signatures that differ between cancer types and have associations with variations in DNA mutational signatures. This compendium of RNA alterations in the genomic context provides a rich resource for identifying genes and mechanisms that are functionally implicated in cancer.
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| 3. |
- Chan, Sherwin, et al.
(författare)
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Regulation of PfEMP1-VAR2CSA translation by a Plasmodium translation-enhancing factor
- 2017
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Ingår i: Nature Microbiology. - : Springer Science and Business Media LLC. - 2058-5276. ; 2:7
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Tidskriftsartikel (refereegranskat)abstract
- Pregnancy-associated malaria commonly involves the binding of Plasmodium falciparum-infected erythrocytes to placental chondroitin sulfate A (CSA) through the PfEMP1-VAR2CSA protein. VAR2CSA is translationally repressed by an upstream open reading frame. In this study, we report that the P. falciparum translation enhancing factor (PTEF) relieves upstream open reading frame repression and thereby facilitates VAR2CSA translation. VAR2CSA protein levels in var2csa-transcribing parasites are dependent on the expression level of PTEF, and the alleviation of upstream open reading frame repression requires the proteolytic processing of PTEF by PfCalpain. Cleavage generates a C-terminal domain that contains a sterile-alpha-motif-like domain. The C-terminal domain is permissive to cytoplasmic shuttling and interacts with ribosomes to facilitate translational derepression of the var2csa coding sequence. It also enhances translation in a heterologous translation system and thus represents the first non-canonical translation enhancing factor to be found in a protozoan. Our results implicate PTEF in regulating placental CSA binding of infected erythrocytes.
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| 4. |
- Cunha, Maria Lucelinda, et al.
(författare)
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Leaching behaviour of industrial oxidic by-products : possibilities to use as neutralisation agent in bioleaching
- 2008
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Ingår i: Advanced Materials Forum IV. - Stafa-Zurich : Trans Tech Publications Inc.. ; , s. 748-752
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Konferensbidrag (refereegranskat)abstract
- In this study chemical leaching with sulphuric acid has been performed on 10 selected oxidic by-products in order to determine their neutralising capacity. The ultimate aim with this work is to replace the lime or limestone normally used in bioleaching operations to maintain pH at 1.5, the optimum pH-level for bioleaching microorganisms, with oxidic by-products. The investigated by-products includes three ashes from combustion for energy production, five slag samples from ore and scrap based steelmaking, an EAF dust and mesa lime from a paper and pulp industry, slaked lime (Ca(OH)2) was used as reference material. The neutralising potential of the by-products were evaluated by leaching them with sulphuric acid and comparing the amount of acid needed to that of the reference. Most of the by-products examined had good neutralisation potential and some had even higher capacities than Ca(OH)2. Neutralisation kinetics were lower for some slag products due to slow dissolution of some of the silicates present, but kinetics are considered good enough since stirred tank bioleaching is a relatively slow process. Zinc recoveries from the zinc containing materials were high, which thus is an additional benefit if these materials were to be used for neutralisation in a bioleaching process for zinc recovery.
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| 5. |
- Cunha, Maria Lucelinda, et al.
(författare)
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Possibilities to use oxidic by-products for precipitation of Fe/As from leaching solutions for subsequent base metal recovery
- 2008
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Ingår i: Minerals Engineering. - : Elsevier BV. - 0892-6875 .- 1872-9444. ; 21:1, s. 38-47
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Tidskriftsartikel (refereegranskat)abstract
- In acidic biological and chemical leaching processes for base metal recovery, iron is dissolved in addition to the desired metal values. Prior to valuable metal extraction iron has to be removed. This is usually achieved through hydroxide precipitation of ferric iron by the addition of lime or limestone to a pH of approximately 3 whereby ferric hydroxide is formed. The aim of this work has been to investigate the possibility to substitute lime or limestone with oxidic industrial by-products for neutralisation and precipitation of iron from leaching solutions. The neutralisation potential for 10 selected oxidic by-products like slags, ashes and dusts were examined and compared with slaked lime.Experiments were performed by decreasing pH to 3 by additions of H2SO4 to slurry of respective by-product at an S/L ratio of 1/10 at 25 °C and continued till no changes in pH were observed during 10 days. Original samples, residues and solutions were analysed by ICP-MS and XRD in order to identify potential harmful elements for the subsequent metal recovery steps.Characterisation of the by-products revealed high concentrations of oxides such as lime, calcite and metal oxides as well as different forms of silicates in the materials which all dissolved at pH 3. The neutralising potential was found to be high for most of the by-products investigated and in the case of Ladle slag it was even higher than for slaked lime. Slags generally had higher neutralisation potential and long-term effects while the ashes had high initial reactivity which is important for continuous neutralisation in stirred tanks with limited retention times. The most reactive materials were Bioash and Mesa lime which both contained considerable amounts of calcite. Replacement of the conventional lime and limestone with oxidic by-products for neutralisation of acidic leaching solutions has the potential to save costs, environmental resources, reduce CO2 emissions and to recycle metal values like zinc contained in the by-products.
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| 6. |
- Degiacomi, Giulia, et al.
(författare)
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Micrococcin P1-A bactericidal thiopeptide active against Mycobacterium tuberculosis
- 2016
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Ingår i: Tuberculosis. - : Elsevier BV. - 1472-9792 .- 1873-281X. ; 100, s. 95-101
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Tidskriftsartikel (refereegranskat)abstract
- The lack of proper treatment for serious infectious diseases due to the emergence of multidrug resistance reinforces the need for the discovery of novel antibiotics. This is particularly true for tuberculosis (TB) for which 3.7% of new cases and 20% of previously treated cases are estimated to be caused by multi-drug resistant strains. In addition, in the case of TB, which claimed 1.5 million lives in 2014, the treatment of the least complicated, drug sensitive cases is lengthy and disagreeable. Therefore, new drugs with novel targets are urgently needed to control resistant Mycobacterium tuberculosis strains. In this manuscript we report the characterization of the thiopeptide micrococcin P1 as an anti-tubercular agent. Our biochemical experiments show that this antibiotic inhibits the elongation step of protein synthesis in mycobacteria. We have further identified micrococcin resistant mutations in the ribosomal protein L11 (RplK); the mutations were located in the proline loop at the N-terminus. Reintroduction of the mutations into a clean genetic background, confirmed that they conferred resistance, while introduction of the wild type RplK allele into resistant strains re-established sensitivity. We also identified a mutation in the 23S rRNA gene. These data, in good agreement with previous structural studies suggest that also in M. tuberculosis micrococcin P1 functions by binding to the cleft between the 23S rRNA and the L11 protein loop, thus interfering with the binding of elongation factors Tu and G (EF-Tu and EF-G) and inhibiting protein translocation.
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| 7. |
- Dinkla, Inez J.T., et al.
(författare)
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Quantifying microorganisms during biooxidation of arsenite and bioleaching of zinc sulfide
- 2013
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Ingår i: Minerals Engineering. - : Elsevier BV. - 0892-6875 .- 1872-9444. ; 48, s. 25-30
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Tidskriftsartikel (refereegranskat)abstract
- The development of molecular tools for the detection and quantification of both species as well as functional traits, aids in a better understanding and control of microbial processes. Presently, these methods can also be used to assess the activity of these organisms or functions, even in complex ecosystems and difficult matrices such as ores and low pH samples. In this paper we present the versatility of one of these tools, Q-PCR, to allow accurate and fast insight in changes in two types of microbial processes representing two ways in which microbes can interact with metals, bioleaching and bioprecipitation. Using the Q-PCR technique it was possible to identify and quantify the thermoacidophilic archaeon Acidianus sp. to be the main microbial strain responsible for biooxidation of arsenite in a low pH reactor. The method was also used to study the dynamics between the iron oxidizing and sulfur oxidizing acidophiles during bioleaching of a zinc concentrate in a batch reactor system and showed that the iron oxidizer Leptospirillum ferriphilum that dominated the starting culture disappeared upon addition of the concentrate. Gradually, bacterial activity was regained starting with growth of sulfur oxidizers and at later stage iron oxidizers started to grow. Molecular analysis can be used to direct research to the relevant organisms involved and concentrate on improving their application (in the arsenite case Acidianus sp.) or in understanding appearances and disappearances of microorganisms (during leaching of zinc concentrate the disappearance of Leptospirillum after high inoculation levels) in order to allow optimization of leaching efficiencies at the lowest (oxygen) costs.
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| 8. |
- Ederth, Josefine, et al.
(författare)
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A single-step method for purification of active His-tagged ribosomes from a genetically engineered Escherichia coli
- 2009
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 37:2, s. e15-
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Tidskriftsartikel (refereegranskat)abstract
- With the rapid development of the ribosome field in recent years a quick, simple and high-throughput method for purification of the bacterial ribosome is in demand. We have designed a new strain of Escherichia coli (JE28) by an in-frame fusion of a nucleotide sequence encoding a hexa-histidine affinity tag at the 3-end of the single copy rplL gene (encoding the ribosomal protein L12) at the chromosomal site of the wild-type strain MG1655. As a result, JE28 produces a homogeneous population of ribosomes (His)(6)-tagged at the C-termini of all four L12 proteins. Furthermore, we have developed a single-step, high-throughput method for purification of tetra-(His)(6)-tagged 70S ribosomes from this strain using affinity chromatography. These ribosomes, when compared with the conventionally purified ones in sucrose gradient centrifugation, 2D-gel, dipeptide formation and a full-length protein synthesis assay showed higher yield and activity. We further describe how this method can be adapted for purification of ribosomal subunits and mutant ribosomes. These methodologies could, in principle, also be used to purify any functional multimeric complex from the bacterial cell.
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| 9. |
- Feng, Boya, et al.
(författare)
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Structural and Functional Insights into the Mode of Action of a Universally Conserved Obg GTPase
- 2014
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Ingår i: PLoS biology. - : Public Library of Science (PLoS). - 1544-9173 .- 1545-7885. ; 12:5, s. e1001866-
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Tidskriftsartikel (refereegranskat)abstract
- Obg proteins are a family of P-loop GTPases, conserved from bacteria to human. The Obg protein in Escherichia coli (ObgE) has been implicated in many diverse cellular functions, with proposed molecular roles in two global processes, ribosome assembly and stringent response. Here, using pre-steady state fast kinetics we demonstrate that ObgE is an anti-association factor, which prevents ribosomal subunit association and downstream steps in translation by binding to the 50S subunit. ObgE is a ribosome dependent GTPase; however, upon binding to guanosine tetraphosphate (ppGpp), the global regulator of stringent response, ObgE exhibits an enhanced interaction with the 50S subunit, resulting in increased equilibrium dissociation of the 70S ribosome into subunits. Furthermore, our cryo-electron microscopy (cryo-EM) structure of the 50S? ObgE? GMPPNP complex indicates that the evolutionarily conserved N-terminal domain (NTD) of ObgE is a tRNA structural mimic, with specific interactions with peptidyl-transferase center, displaying a marked resemblance to Class I release factors. These structural data might define ObgE as a specialized translation factor related to stress responses, and provide a framework towards future elucidation of functional interplay between ObgE and ribosome-associated (p) ppGpp regulators. Together with published data, our results suggest that ObgE might act as a checkpoint in final stages of the 50S subunit assembly under normal growth conditions. And more importantly, ObgE, as a (p) ppGpp effector, might also have a regulatory role in the production of the 50S subunit and its participation in translation under certain stressed conditions. Thus, our findings might have uncovered an under-recognized mechanism of translation control by environmental cues.
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| 10. |
- Fislage, Marcus, et al.
(författare)
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Cryo-EM shows stages of initial codon selection on the ribosome by aa-tRNA in ternary complex with GTP and the GTPase-deficient EF-Tu(H84A)
- 2018
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Ingår i: Nucleic Acids Research. - : OXFORD UNIV PRESS. - 0305-1048 .- 1362-4962. ; 46:11, s. 5861-5874
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Tidskriftsartikel (refereegranskat)abstract
- The GTPase EF-Tu in ternary complex with GTP and aminoacyl-tRNA (aa-tRNA) promotes rapid and accurate delivery of cognate aa-tRNAs to the ribosomal A site. Here we used cryo-EM to study the molecular origins of the accuracy of ribosome-aided recognition of a cognate ternary complex and the accuracy-amplifying role of themonitoring bases A1492, A1493 and G530 of the 16S rRNA. We used the GTPase-deficient EF-Tu variant H84A with native GTP, rather than non-cleavable GTP analogues, to trap a near-cognate ternary complex in high-resolution ribosomal complexes of varying codon-recognition accuracy. We found that ribosome complexes trapped by GTPase-deficicent ternary complex due to the presence of EF-TuH84A or non-cleavable GTP analogues have very similar structures. We further discuss speed and accuracy of initial aa-tRNA selection in terms of conformational changes of aa-tRNA and stepwise activation of the monitoring bases at the decoding center of the ribosome.
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