scholarly journals Is the In Situ Accessibility of the 16S rRNA of Escherichia coli for Cy3-Labeled Oligonucleotide Probes Predicted by a Three-Dimensional Structure Model of the 30S Ribosomal Subunit?

2003 ◽  
Vol 69 (8) ◽  
pp. 4935-4941 ◽  
Author(s):  
Sebastian Behrens ◽  
Bernhard M. Fuchs ◽  
Florian Mueller ◽  
Rudolf Amann
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
Rational Design ◽  
Tertiary Structure ◽  
Sodium Dodecyl ◽  
Three Dimensional ◽  
Ribosomal Subunit ◽  
Order Structure ◽  
Structure Model

ABSTRACT Systematic studies on the hybridization of fluorescently labeled, rRNA-targeted oligonucleotides have shown strong variations in in situ accessibility. Reliable predictions of target site accessibility would contribute to more-rational design of probes for the identification of individual microbial cells in their natural environments. During the past 3 years, numerous studies of the higher-order structure of the ribosome have advanced our understanding of its spatial conformation. These studies range from the identification of rRNA-rRNA interactions based on covariation analyses to physical imaging of the ribosome for the identification of protein-rRNA interactions. Here we reevaluate our Escherichia coli 16S rRNA in situ accessibility data with regard to a tertiary-structure model of the small subunit of the ribosome. We localized target sequences of 176 oligonucleotides on a 3.0-Å-resolution three-dimensional (3D) model of the 30S ribosomal subunit. Little correlation was found between probe hybridization efficiency and the proximity of the probe target region to the surface of the 30S ribosomal subunit model. We attribute this to the fact that fluorescence in situ hybridization is performed on fixed cells containing denatured ribosomes, whereas 3D models of the ribosome are based on its native conformation. The effects of different fixation and hybridization protocols on the fluorescence signals conferred by a set of 10 representative probes were tested. The presence or absence of the strongly denaturing detergent sodium dodecyl sulfate had a much more pronounced effect than a change of fixative from paraformaldehyde to ethanol.

scholarly journals In Situ Accessibility of Escherichia coli 23S rRNA to Fluorescently Labeled Oligonucleotide Probes

2001 ◽  
Vol 67 (2) ◽  
pp. 961-968 ◽  
Author(s):  
Bernhard M. Fuchs ◽  
Kazuaki Syutsubo ◽  
Wolfgang Ludwig ◽  
Rudolf Amann
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
23S Rrna ◽  
Order Structure ◽  
Oligonucleotide Probes ◽  
Class Iii ◽  
Resolution Analysis ◽  
Identification Of Bacteria ◽  
Fine Resolution

ABSTRACT One of the main causes of failure of fluorescence in situ hybridization with rRNA-targeted oligonucleotides, besides low cellular ribosome content and impermeability of cell walls, is the inaccessibility of probe target sites due to higher-order structure of the ribosome. Analogous to a study on the 16S rRNA (B. M. Fuchs, G. Wallner, W. Beisker, I. Schwippl, W. Ludwig, and R. Amann, Appl. Environ. Microbiol. 64:4973–4982, 1998), the accessibility of the 23S rRNA of Escherichia coli DSM 30083T was studied in detail with a set of 184 CY3-labeled oligonucleotide probes. The probe-conferred fluorescence was quantified flow cytometrically. The brightest signal resulted from probe 23S-2018, complementary to positions 2018 to 2035. The distribution of probe-conferred cell fluorescence in six arbitrarily set brightness classes (classes I to VI, 100 to 81%, 80 to 61%, 60 to 41%, 40 to 21%, 20 to 6%, and 5 to 0% of the brightness of 23S-2018, respectively) was as follows: class I, 3%; class II, 21%; class III, 35%; class IV, 18%; class V, 16%; and class VI, 7%. A fine-resolution analysis of selected areas confirmed steep changes in accessibility on the 23S RNA to oligonucleotide probes. This is similar to the situation for the 16S rRNA. Indeed, no significant differences were found between the hybridization of oligonucleotide probes to 16S and 23S rRNA. Interestingly, indications were obtained of an effect of the type of fluorescent dye coupled to a probe on in situ accessibility. The results were translated into an accessibility map for the 23S rRNA ofE. coli, which may be extrapolated to other bacteria. Thereby, it may contribute to a better exploitation of the high potential of the 23S rRNA for identification of bacteria in the future.

scholarly journals Making All Parts of the 16S rRNA of Escherichia coli Accessible In Situ to Single DNA Oligonucleotides

2006 ◽  
Vol 72 (1) ◽  
pp. 733-744 ◽  
Author(s):  
L. Şafak Yilmaz ◽  
Hatice E. Ökten ◽  
Daniel R. Noguera
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
Fluorescence Intensity ◽  
Rational Design ◽  
Mechanistic Model ◽  
Mechanistic Modeling ◽  
E Coli ◽  
Dna Oligonucleotides ◽  
Nucleic Acid Interactions

ABSTRACT rRNA accessibility is a major sensitivity issue limiting the design of working probes for fluorescence in situ hybridization (FISH). Previous studies empirically highlighted the accessibility of target sites on rRNA maps by grouping probes into six classes according to their brightness levels. In this study, a recently proposed mechanistic model of FISH, based on the thermodynamics of secondary nucleic acid interactions, was used to evaluate the accessibility of the 16S rRNA of Escherichia coli to fluorescein-labeled oligonucleotides when thermodynamic and kinetic barriers were eliminated. To cover the entire 16S rRNA, 109 probes were designed with an average thermodynamic affinity (ΔG o overall) of −13.5 kcal/mol. Fluorescence intensity was measured by flow cytometry, and a brightness threshold between classes 3 and 4 was used as the requirement for proof of accessibility. While 46% of the probes were above this threshold with conventional 3-h hybridizations, extending the incubation period to 96 h dramatically increased the fraction of bright probes to 86%. Insufficient thermodynamic affinity and/or fluorophore quenching was demonstrated to cause the low fluorescence intensity of the remaining 14% of the probes. In the end, it was proven that every nucleotide in the 16S rRNA of E. coli could be targeted with a bright probe and, therefore, that there were no truly inaccessible target regions in the 16S rRNA. Based on our findings and mechanistic modeling, a rational design strategy involving ΔG o overall, hybridization kinetics, and fluorophore quenching is recommended for the development of bright probes.

Structure of ribosomes

1979 ◽  
Vol 57 (11) ◽  
pp. 1251-1261 ◽  
Author(s):  
H. G. Wittmann
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Proteins ◽  
Tertiary Structure ◽  
Protein Biosynthesis ◽  
Ribosomal Subunit ◽  
Immunological Methods ◽  
Detailed Knowledge ◽  
Essential Difference ◽  
E Coli

Ribosomes are multicomponent particles on which biosynthesis of proteins occurs in all organisms. The best known ribosome, namely that of Escherichia coli, consists of three RNAs and 53 different proteins. All proteins have been isolated and characterized by chemical, physical, and immunological methods. The primary sequences of 47 E. coli ribosomal proteins have so far been determined. Studies of the shape, as well as the secondary and tertiary structure, of the proteins are in progress.Various techniques (e.g. immune electron microscopy and cross-linking of neighbouring components in situ) give information about the architecture of the ribosomal particle. The first technique resulted in illustrative and detailed knowledge not only on the shape of the ribosomal subunits but also about the location of many proteins on the surface of the particles. The analysis of cross-links between ribosomal proteins and (or) RNAs has in several cases been pursued to the level of elucidating which amino acids and (or) nucleotides are cross-linked together in situ. Reconstitution of a fully active E. coli 50S ribosomal subunit from its isolated RNA and protein components can be accomplished by means of a two-step incubation procedure. From the analysis of the intermediates occurring during the reconstitution process it has been concluded that the in vitro reconstitution process resembles the in vivo assembly of 50S subunits in many respects. Escherichia coli mutants with alterations in almost all ribosomal proteins have been isolated. Their biochemical and genetic analyses are very useful tools for obtaining information about the structure, function, and biosynthesis of ribosomes as well as about the location of the genes for these proteins on the chromosome. From comparative electrophoretic, immunological, protein–chemical, and reconstitution studies on ribosomes from various species it has become clear that there is little homology between ribosomes from prokaryotes and those from eukaryotes. This finding is surprising since there is no essential difference in the way in which pro- and eu-karyotic ribosomes function in protein biosynthesis.

Structural organization of escherichia coli ribosomes as determined by immuno electron microscopy

1978 ◽  
Vol 36 (2) ◽  
pp. 166-167 ◽  
Author(s):  
G. Stöffler ◽  
R.W. Bald ◽  
J. Dieckhoff ◽  
H. Eckhard ◽  
R. Lührmann ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Escherichia Coli ◽  
Ribosomal Proteins ◽  
Structural Organization ◽  
Three Dimensional ◽  
Ribosomal Subunit ◽  
Spatial Arrangement ◽  
Small Subunit ◽  
Antibody Binding ◽  
Immuno Electron Microscopy

A central step towards an understanding of the structure and function of the Escherichia coli ribosome, a large multicomponent assembly, is the elucidation of the spatial arrangement of its 54 proteins and its three rRNA molecules. The structural organization of ribosomal components has been investigated by a number of experimental approaches. Specific antibodies directed against each of the 54 ribosomal proteins of Escherichia coli have been performed to examine antibody-subunit complexes by electron microscopy. The position of the bound antibody, specific for a particular protein, can be determined; it indicates the location of the corresponding protein on the ribosomal surface.The three-dimensional distribution of each of the 21 small subunit proteins on the ribosomal surface has been determined by immuno electron microscopy: the 21 proteins have been found exposed with altogether 43 antibody binding sites. Each one of 12 proteins showed antibody binding at remote positions on the subunit surface, indicating highly extended conformations of the proteins concerned within the 30S ribosomal subunit; the remaining proteins are, however, not necessarily globular in shape (Fig. 1).

scholarly journals Construction of A Preliminary Three-Dimensional Structure Simian betaretrovirus Serotype-2 (SRV-2) Reverse Transcriptase Isolated from Indonesian Cynomolgus Monkey

2020 ◽  
Vol 31 (3) ◽  
pp. 47-61
Author(s):  
Uus Saepuloh ◽  
Diah Iskandriati ◽  
Joko Pamungkas ◽  
Dedy Duryadi Solihin ◽  
Sela Septima Mariya ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Cynomolgus Monkey ◽  
Tertiary Structure ◽  
Vaccine Development ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Nucleotide Position ◽  
Structure Model ◽  
Three Dimensional Structure ◽  
Hiv 1

Simian betaretrovirus serotype-2 (SRV-2) is an important pathogenic agent in Asian macaques. It is a potential confounding variable in biomedical research. SRV-2 also provides a valuable viral model compared to other retroviruses which can be used for understanding many aspects of retroviral-host interactions and immunosuppression, infection mechanism, retroviral structure, antiretroviral and vaccine development. In this study, we isolated the gene encoding reverse transcriptase enzyme (RT) of SRV-2 that infected Indonesian cynomolgus monkey (Mf ET1006) and predicted the three dimensional structure model using the iterative threading assembly refinement (I-TASSER) computational programme. This SRV-2 RT Mf ET1006 consisted of 547 amino acids at nucleotide position 3284–4925 of whole genome SRV-2. The polymerase active site located in the finger/palm subdomain characterised by three conserved catalytic aspartates (Asp90, Asp165, Asp166), and has a highly conserved YMDD motif as Tyr163, Met164, Asp165 and Asp166. We estimated that this SRV-2 RT Mf ET1006 structure has the accuracy of template modelling score (TM-score 0.90 ± 0.06) and root mean square deviation (RMSD) 4.7 ± 3.1Å, indicating that this model can be trusted and the accuracy can be seen from the appearance of protein folding in tertiary structure. The superpositionings between SRV-2 RT Mf ET1006 and Human Immunodeficiency Virus-1 (HIV-1) RT were performed to predict the structural in details and to optimise the best fits for illustrations. This SRV-2 RT Mf ET1006 structure model has the highest homology to HIV-1 RT (2B6A.pdb) with estimated accuracy at TM-score 0.911, RMSD 1.85 Å, and coverage of 0.953. This preliminary study of SRV-2 RT Mf ET1006 structure modelling is intriguing and provide some information to explore the molecular characteristic and biochemical mechanism of this enzyme.

scholarly journals In situ delivery of biobutyrate by probiotic Escherichia coli for cancer therapy

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chung-Jen Chiang ◽  
Yan-Hong Hong
Keyword(s):  
Escherichia Coli ◽  
Cancer Therapy ◽  
Rational Design ◽  
Hypoxic Condition ◽  
Human Colorectal Cancer ◽  
Apoptosis Pathway ◽  
Mitochondrial Apoptosis Pathway ◽  
Cycle Arrest ◽  
Human Colorectal Cancer Cell

AbstractButyrate has a bioactive function to reduce carcinogenesis. To achieve targeted cancer therapy, this study developed bacterial cancer therapy (BCT) with butyrate as a payload. By metabolic engineering, Escherichia coli Nissle 1917 (EcN) was reprogrammed to synthesize butyrate (referred to as biobutyrate) and designated EcN-BUT. The adopted strategy includes construction of a synthetic pathway for biobutyrate and the rational design of central metabolism to increase the production of biobutyrate at the expense of acetate. With glucose, EcN-BUT produced primarily biobutyrate under the hypoxic condition. Furthermore, human colorectal cancer cell was administrated with the produced biobutyrate. It caused the cell cycle arrest at the G1 phase and induced the mitochondrial apoptosis pathway independent of p53. In the tumor-bearing mice, the injected EcN-BUT exhibited tumor-specific colonization and significantly reduced the tumor volume by 70%. Overall, this study opens a new avenue for BCT based on biobutyrate.

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