Imaging of ribosomal RNA-protein interactions by dark-field STEM

1989 ◽  
Vol 47 ◽  
pp. 250-251
Author(s):  
M. Boublik ◽  
V. Mandiyan ◽  
S. Tumminia ◽  
J.F. Hainfeld ◽  
J.S. Wall
Keyword(s):  
Nucleic Acid ◽  
16S Rrna ◽  
Dark Field ◽  
Radius Of Gyration ◽  
Central Domain ◽  
The Core ◽  
16S Rna ◽  
30S Subunit ◽  
Core Particles

Success in protein-free deposition of native nucleic acid molecules from solutions of selected ionic conditions prompted attempts for high resolution imaging of nucleic acid interactions with proteins, not attainable by conventional EM. Since the nucleic acid molecules can be visualized in the dark-field STEM mode without contrasting by heavy atoms, the established linearity between scattering cross-section and molecular weight can be applied to the determination of their molecular mass (M) linear density (M/L), mass distribution and radius of gyration (RG). Determination of these parameters promotes electron microscopic imaging of biological macromolecules by STEM to a quantitative analytical level. This technique is applied to study the mechanism of 16S rRNA folding during the assembly process of the 30S ribosomal subunit of E. coli. The sequential addition of protein S4 which binds to the 5'end of the 16S rRNA and S8 and S15 which bind to the central domain of the molecule leads to a corresponding increase of mass and increased coiling of the 16S rRNA in the core particles. This increased compactness is evident from the decrease in RG values from 114Å to 91Å (in “ribosomal” buffer consisting of 10 mM Hepes pH 7.6, 60 mM KCl, 2 m Mg(OAc)2, 1 mM DTT). The binding of S20, S17 and S7 which interact with the 5'domain, the central domain and the 3'domain, respectively, continues the trend of mass increase. However, the RG values of the core particles exhibit a reverse trend, an increase to 108Å. In addition, the binding of S7 leads to the formation of a globular mass cluster with a diameter of about 115Å and a mass of ∽300 kDa. The rest of the mass, about 330 kDa, remains loosely coiled giving the particle a “medusa-like” appearance. These results provide direct evidence that 16S RNA undergoes significant structural reorganization during the 30S subunit assembly and show that its interactions with the six primary binding proteins are not sufficient for 16S rRNA coiling into particles resembling the native 30S subunit, contrary to what has been reported in the literature.

Protein-induced conformational changes in 16S rRNA during assembly of the Escherichia Coli 30S ribosomal subunit: A STEM study

1987 ◽  
Vol 45 ◽  
pp. 910-911
Author(s):  
M. Boublik ◽  
V. Mandiyan ◽  
J.F. Hainfeld ◽  
J.S. Wall
Keyword(s):  
16S Rrna ◽  
Conformational Changes ◽  
Ribosomal Proteins ◽  
Structural Changes ◽  
Ribosomal Subunit ◽  
Compact Structure ◽  
E Coli ◽  
Freeze Dried ◽  
16S Rna ◽  
30S Subunit

The aim of this study is to understand the mechanism of 16S rRNA folding into the compact structure of the small 30S subunit of E. coli ribosome. The assembly of the 30S E. coli ribosomal subunit is a sequence of specific interactions of 16S rRNA with 21 ribosomal proteins (S1-S21). Using dedicated high resolution STEM we have monitored structural changes induced in 16S rRNA by the proteins S4, S8, S15 and S20 which are involved in the initial steps of 30S subunit assembly. S4 is the first protein to bind directly and stoichiometrically to 16S rRNA. Direct binding also occurs individually between 16S RNA and S8 and S15. However, binding of S20 requires the presence of S4 and S8. The RNA-protein complexes are prepared by the standard reconstitution procedure, dialyzed against 60 mM KCl, 2 mM Mg(OAc)2, 10 mM-Hepes-KOH pH 7.5 (Buffer A), freeze-dried and observed unstained in dark field at -160°.

scholarly journals Shape changes and cooperativity in the folding of the central domain of the 16S ribosomal RNA

2021 ◽  
Vol 118 (10) ◽  
pp. e2020837118
Author(s):  
Naoto Hori ◽  
Natalia A. Denesyuk ◽  
D. Thirumalai
Keyword(s):  
16S Rrna ◽  
Ribosomal Proteins ◽  
Quantitative Description ◽  
Small Subunit ◽  
Coarse Grained ◽  
Radius Of Gyration ◽  
Central Domain ◽  
Induced Folding ◽  
Starting Point ◽  
Hydroxyl Radical Footprinting

Both the small and large subunits of the ribosome, the molecular machine that synthesizes proteins, are complexes of ribosomal RNAs (rRNAs) and a number of proteins. In bacteria, the small subunit has a single 16S rRNA whose folding is the first step in its assembly. The central domain of the 16S rRNA folds independently, driven either by Mg2+ ions or by interaction with ribosomal proteins. To provide a quantitative description of ion-induced folding of the ∼350-nucleotide rRNA, we carried out extensive coarse-grained molecular simulations spanning Mg2+ concentration between 0 and 30 mM. The Mg2+ dependence of the radius of gyration shows that globally the rRNA folds cooperatively. Surprisingly, various structural elements order at different Mg2+ concentrations, indicative of the heterogeneous assembly even within a single domain of the rRNA. Binding of Mg2+ ions is highly specific, with successive ion condensation resulting in nucleation of tertiary structures. We also predict the Mg2+-dependent protection factors, measurable in hydroxyl radical footprinting experiments, which corroborate the specificity of Mg2+-induced folding. The simulations, which agree quantitatively with several experiments on the folding of a three-way junction, show that its folding is preceded by formation of other tertiary contacts in the central junction. Our work provides a starting point in simulating the early events in the assembly of the small subunit of the ribosome.

scholarly journals Shape changes and cooperativity in the folding of central domain of the 16S ribosomal RNA

2020 ◽  
Author(s):  
Naoto Hori ◽  
Natalia A Denesyuk ◽  
D Thirumalai
Keyword(s):  
16S Rrna ◽  
Ribosomal Proteins ◽  
Quantitative Description ◽  
Small Subunit ◽  
Coarse Grained ◽  
Radius Of Gyration ◽  
Central Domain ◽  
Induced Folding ◽  
Starting Point ◽  
Hydroxyl Radical Footprinting

Both the small and large subunits of the ribosome, the molecular machine that synthesizes proteins, are complexes of ribosomal RNAs (rRNAs) and a number of proteins. In bacteria, the small subunit has a single 16S rRNA whose folding is the first step in its assembly. The central domain of the 16S rRNA folds independently, driven either by Mg2+ions or by interaction with ribosomal proteins. In order to provide a quantitative description of ion-induced folding of the ~350 nucleotide rRNA, we carried out extensive coarse-grained molecular simulations spanning Mg2+concentration between 0−30 mM. The Mg2+dependence of the radius of gyration shows that globally the rRNA folds cooperatively. Surprisingly, various structural elements order at different Mg2+concentrations, indicative of the heterogeneous assembly even within a single domain of the rRNA. Binding of Mg2+ions is highly specific, with successive ion condensation resulting in nucleation of tertiary structures. We also predict the Mg2+-dependent protection factors, measurable in hydroxyl radical footprinting experiments, which corroborate the specificity of Mg2+-induced folding. The simulations, which agree quantitatively with several experiments on the folding of a three-way junction, show that its folding is preceded by formation of other tertiary contacts in the central junction. Our work provides a starting point in simulating the early events in the assembly of the small subunit of the ribosome.

The many faces of STEM in materials science

1991 ◽  
Vol 49 ◽  
pp. 440-441
Author(s):  
John Silcox
Keyword(s):  
Materials Science ◽  
Auger Spectroscopy ◽  
Dark Field ◽  
High Tc Superconductors ◽  
High Tc ◽  
Acoustic Environment ◽  
Electromagnetic Vibration ◽  
The Many ◽  
Serial Mode

Determination of the microstructure and microchemistry of small features often provides the insight needed for the understanding of processes in real materials. In many cases, it is not adequate to use microscopy alone. Microdiffraction and microspectroscopic information such as EELS, X-ray microprobe analysis and Auger spectroscopy can all contribute vital parts of the picture. For a number of reasons, dedicated STEM offers considerable promise as a quantitative instrument. In this paper, we review progress towards effective quantitative use of STEM with illustrations drawn from studies of high Tc superconductors, compound semiconductors and metallization of H-terminated silicon.Intrinsically, STEM is a quantitative instrument. Images are acquired directly by detectors in serial mode which is particularly convenient for digital image acquisition, control and display. The VG HB501A at Cornell has been installed in a particularly stable electromagnetic, vibration and acoustic environment. Care has been paid to achieving UHV conditions (i.e., 10-10 Torr). Finally, it has been interfaced with a VAX 3200 work station by Kirkland. This permits, for example, the acquisition of bright field (or energy loss) images and dark field images simultaneously as quantitative arrays in perfect registration.

Application of Ogur & Rosen's Method for the Determination of Nucleic Acid Content of Bacteria

1957 ◽  
Vol 12 (2) ◽  
pp. 125-129 ◽  
Author(s):  
Nobuyasu KAWASAKI ◽  
Ichiro TAKI ◽  
Chiaki WATANABE ◽  
Kiyoshi MATOBA ◽  
Mokichiro NISHIO ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Acid Content ◽  
Nucleic Acid Content

Nauwkeurige methode voor de aanwasbepaling van het grondvlak met behulp van de Pressler-boor

1968 ◽  
Vol 12 ◽  
Author(s):  
R. Goossens
Keyword(s):  
Basal Area ◽  
Precise Determination ◽  
Maximum Diameter ◽  
Precise Method ◽  
Maximum Radius ◽  
The Core ◽  
Two Parameters

A precise method for the determination of the increment of the  basal area using the PressIer bore. Refering to  previous research showing that the basal area of the corsica pine could be  characterized by an ellips, we present in this paper a precise method for the  determination of the increment of the basal area. In this method we determine  the direction of the maximum diameter, we measure this diameter and we take a  core in one of the points of tangency of the caliper with the measured tree.  The determination of the diameter perpendicular to the maximum diameter  finishes the work wich is to be done in the forest. From the classical  measurements effectuated on the core and from the measured diameters we can  then determine the form (V) and the excentricity (e). Substituting these two  parameters in the formula 2 or 2', we can also calculate the error of a  radius measured on the core with respect to the representative radius, This  error with them allow us to correct the measured value of the minimum or the  maximum radius and we will be able to do a precise determination of the  increment.

scholarly journals Multiplexed Plasmonic Nano-Labeling for Bioimaging of Cytological Stained Samples

Cancers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 3509
Author(s):  
Paule Marcoux-Valiquette ◽  
Cécile Darviot ◽  
Lu Wang ◽  
Andrée-Anne Grosset ◽  
Morteza Hasanzadeh Kafshgari ◽  
...  
Keyword(s):  
Accurate Determination ◽  
Cell Types ◽  
Fine Needle Biopsy ◽  
Dark Field ◽  
Imaging Method ◽  
New Methods ◽  
Formalin Fixed Paraffin ◽  
Dark Field Microscopy ◽  
Formalin Fixed Paraffin Embedded

Reliable cytopathological diagnosis requires new methods and approaches for the rapid and accurate determination of all cell types. This is especially important when the number of cells is limited, such as in the cytological samples of fine-needle biopsy. Immunoplasmonic-multiplexed- labeling may be one of the emerging solutions to such problems. However, to be accepted and used by the practicing pathologists, new methods must be compatible and complementary with existing cytopathology approaches where counterstaining is central to the correct interpretation of immunolabeling. In addition, the optical detection and imaging setup for immunoplasmonic-multiplexed-labeling must be implemented on the same cytopathological microscope, not interfere with standard H&E imaging, and operate as a second easy-to-use imaging method. In this article, we present multiplex imaging of four types of nanoplasmonic markers on two types of H&E-stained cytological specimens (formalin-fixed paraffin embedded and non-embedded adherent cancer cells) using a specially designed adapter for SI dark-field microscopy. The obtained results confirm the effectiveness of the proposed optical method for quantitative and multiplex identification of various plasmonic NPs, and the possibility of using immunoplasmonic-multiplexed-labeling for cytopathological diagnostics.

scholarly journals nanocrystals (0 ≤ x ≤ 1): growth, size control and shell formation on β-NaCeF4:Tb core particles

CrystEngComm ◽  
2020 ◽  
Vol 22 (46) ◽  
pp. 8036-8044
Author(s):  
Jannis Wehmeier ◽  
Markus Haase
Keyword(s):  
Size Control ◽  
Lattice Parameters ◽  
Core Material ◽  
Shell Formation ◽  
Shell Material ◽  
Strontium Content ◽  
The Core ◽  
Lighter Lanthanides ◽  
Core Particles

is an interesting shell material for β-NaREF4 particles of the lighter lanthanides (RE = Ce, Pr, Nd), as variation of its strontium content x allows to vary its lattice parameters and match those of the core material.

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