Struktur von funktionellen Komplexen der kleinen ribosomalen Untereinheit

0\. TITLE Table of contents 1\. INTRODUCTION 1 1.1 The Ribosome 1 1.2 Antibiotics acting on the ribosome 5 1.3 X-ray crystallography 6 1.4 Crystallographic studies of the ribosome 8 1.5 Thermus thermophilus organism 9 2\. MATERIALS 10 2.1 Chemicals, Enzymes and kits 10 2.2 Bacterial Strains 11 2.3 Plasmids 11 2.4 Media 11 2.5 Oligodeoxyribonucleotides 11 2.6 Purification columns 12 3\. METHODS 13 3.1 General methods 13 3.2 DNA methods 14 3.3 Ribosome methods 17 3.4 c-DNA methods2 20 3.5 Protein methods 22 3.6 Immunological methods 28 3.7 Crystallographic methods 30 4\. RESULTS 33 4.1 c-DNA 33 4.2 The Initiation Factor 3 42 4.3 Structure determination 54 5\. DISCUSSION 65 5.1 c-DNA 65 5.2 The Initiation Factor 3 66 5.3 Antibiotics 67 6\. REFERENCES 71 7\. APPENDIX 82The small ribosomal subunit is responsible for the decoding of the genetic information and plays a key role in the initiation of protein synthesis. To elucidate the mechanism of action of the ribosome and of the initiation process, we focused on the determination of the three dimensional structure by X-ray crystallography of the 30S subunit in complex with different short oligonucleotides complementing specific regions of the 16S rRNA, the initiation factor 3 and two antibiotics, edeine and tetracycline. We developed a new method to label the 30S subunit of T. thermophilus with oligonucleotides complementary to the 16S rRNA. In this way we could localise the 3' end of the 16S rRNA, which contains the anti-Shine-Dalgarno (anti-SD) sequence. We localised IF3-C at the solvent side of the platform, close to the anti-SD region of the 16S rRNA. The position of IF3-C shows clearly that the anti- association activity of IF3 is not due to the physical blockage of the inter- subunit interface, but it is rather the product of a change in the conformational dynamics of the subunit. We localised edeine in the vicinity of the E-site, interacting with universally conserved nucleotides in Helices 24 (H24), H28, H44 and H45. Our structure offers a good explanation as to why edeine blocks the path of the mRNA between the decoding region and the anti-SD region of the 16S rRNA in prokaryotes. We identified six tetracycline-binding sites on the 30S subunit (Tet-1 -Tet-6). Our data for the six positions of tetracycline can well explain the sometimes contradictory reported biochemical and functional data for tetracycline binding to the 30S subunits.Für die Dekodierung der genetischen Information ist die kleine ribosomale Untereinheit zuständig, die eine Schlüsselrolle bei der Initiation der Proteinsynthese spielt. Um die Vorgänge im Ribosom und während des Initationsprozesses besser verstehen zu können, haben wir das Hauptaugenmerk unserer Untersuchung auf die Bestimmung der dreidimensionalen Struktur der 30S Untereinheit gelegt, die sich im Verbund mit verschiedenen Liganden befindet. Zu diesen Liganden gehören kurze Oligonukleotide, die bestimmte Regionen der 16S rRNA komplementieren, wie der Initiationsfaktor 3 und die Antibiotika Edein und Tetrazyklin. Die Lokalisierung der verschiedenen Liganden wurde mit Hilfe der kristallographischen Röntgenstrukturanalyse durchgeführt. Für unsere Untersuchung haben wir eine neue Methode entwickelt die 30S Untereinheit von T. thermophilus zu markieren. Der 16S rRNA werden komplementäre Oligonukleotide hinzugefügt. Auf diese Weise gelang es uns, das 3'-Ende der 16S rRNA zu lokalisieren, welches die Anti-Shine-Dalgarno (anti-SD) Sequenz enthält. Wir lokalisierten IF3-C auf der Außenseite der Plattform, nahe der anti-SD Region der 16S rRNA. Die Lage von IF3-C macht deutlich, daß die Anti- Assoziationsaktivität von IF3 nicht Folge einer physikalischen Blockierung der "inter-subunit interface" ist, sondern eher durch einen Wechsel in der Konformationsdynamik der Untereinheit hervorgerufen wurde. Wir lokalisierten Edein in der Nachbarschaft der E-Stelle. Es befindet sich in der Interaktion mit universell konservierten Nukleotiden der 16S rRNA in den Helices 24 (H24), H28, H44 und H45. Unser Strukturmodell liefert somit eine gute Erklärung, weshalb Edein den Weg der mRNA zwischen der Dekodierungsregion und der Anti- SD- Region der 16S rRNA in Prokaryonten blockieren kann. Wir identifizierten sechs Tetrazyklin-Bindestellen auf der 30S Untereinheit (Tet-1 bis Tet-6). Die von uns ermittelten Daten, zu den sechs verschiedenen Positionen von Tetrazyklin, erklären in einleuchtender Weise, wie es zu den oft widersprüchlichen Ergebnissen biochemischer und funktionaler Analysen des an die 30S Untereinheiten gebundenen Tetrazyklins kommen konnte

Aportes para una clínica psocanalítica con niños

A partir de una propuesta más abarcativa que se plantea investigar la obra teórica y los relatos clínicos de los analistas que fundaron líneas de trabajo en el campo de la clínica con niños, para rastrear en ellos los modelos de dirección de la cura que se proponen, así como los criterios éticos que se ponen en juego para orientar la labor terapéutica, este trabajo pretende dar cuenta de las ideas principales de Melanie Klein. Para ordenar nuestra exposición la hemos organizado en torno a dos ejes conceptuales: inconsciente/interpretación y transferencia/fin de análisis.Fil: Martinez, Horacio. Facultad de Psicología. Universidad Nacional de Mar del Plata; ArgentinaFil: Cacciari, Analía. Facultad de Psicología. Universidad Nacional de Mar del Plata; ArgentinaFil: Dimov, Marta. Facultad de Psicología. Universidad Nacional de Mar del Plata; ArgentinaFil: Dominguez, Mariana. Facultad de Psicología. Universidad Nacional de Mar del Plata; ArgentinaFil: Krauss, Silvia. Facultad de Psicología. Universidad Nacional de Mar del Plata; ArgentinaFil: Pioletti, Paula. Facultad de Psicología. Universidad Nacional de Mar del Plata; Argentin

Aportes para una clínica psocanalítica con niños

A partir de una propuesta más abarcativa que se plantea investigar la obra teórica y los relatos clínicos de los analistas que fundaron líneas de trabajo en el campo de la clínica con niños, para rastrear en ellos los modelos de dirección de la cura que se proponen, así como los criterios éticos que se ponen en juego para orientar la labor terapéutica, este trabajo pretende dar cuenta de las ideas principales de Melanie Klein. Para ordenar nuestra exposición la hemos organizado en torno a dos ejes conceptuales: inconsciente/interpretación y transferencia/fin de análisis.Fil: Martinez, Horacio. Facultad de Psicología. Universidad Nacional de Mar del Plata; ArgentinaFil: Cacciari, Analía. Facultad de Psicología. Universidad Nacional de Mar del Plata; ArgentinaFil: Dimov, Marta. Facultad de Psicología. Universidad Nacional de Mar del Plata; ArgentinaFil: Dominguez, Mariana. Facultad de Psicología. Universidad Nacional de Mar del Plata; ArgentinaFil: Krauss, Silvia. Facultad de Psicología. Universidad Nacional de Mar del Plata; ArgentinaFil: Pioletti, Paula. Facultad de Psicología. Universidad Nacional de Mar del Plata; Argentin

Crystal structures of complexes of the small ribosomal subunit with tetracycline, edeine and IF3

The small ribosomal subunit is responsible for the decoding of genetic information and plays a key role in the initiation of protein synthesis. We analyzed by X-ray crystallography the structures of three different complexes of the small ribosomal subunit of Thermus thermophilus with the A-site inhibitor tetracycline, the universal initiation inhibitor edeine and the C-terminal domain of the translation initiation factor IF3. The crystal structure analysis of the complex with tetracycline revealed the functionally important site responsible for the blockage of the A-site. Five additional tetracycline sites resolve most of the controversial biochemical data on the location of tetracycline. The interaction of edeine with the small subunit indicates its role in inhibiting initiation and shows its involvement with P-site tRNA. The location of the C-terminal domain of IF3, at the solvent side of the platform, sheds light on the formation of the initiation complex, and implies that the anti-association activity of IF3 is due to its influence on the conformational dynamics of the small ribosomal subunit

Metal Compounds as Tools for the Construction and the Interpretation of Medium-Resolution Maps of Ribosomal Particles

Procedures were developed exploiting organometallic clusters and coordination compounds in combination with heavy metal salts for derivatization of ribosomal crystals. These enabled the construction of multiple isomorphous replacement (MIR) and multiple isomorphous replacement combined with anomalous scattering medium-resolution electron density maps for the ribosomal particles that yield the crystals diffracting to the highest resolution, 3 Å, of the large subunit from Haloarcula marismortui and the small subunit from Thermus thermophilus. The first steps in the interpretation of the 7.3-Å MIR map of the small subunit were made with the aid of a tetrairidium cluster that was covalently attached to exposed sulfhydryls on the particle's surface prior to crystallization. The positions of these sulfhydryls were localized in difference Fourier maps that were constructed with the MIR phases

The identification of selected components in electron density maps of prokaryotic ribosomes at 7 Å resolution

Crystals of small and large ribosomal subunits from thermophilic and halophilic bacteria, diffracting to 3 Å, are being subjected to structural analysis with synchrotron radiation. The bright beam necessary for detecting and collecting the diffraction at the higher-resolution shell causes significant decay even at 25 K. Nevertheless, data collected from native and heavy-atom-derivatized crystals led to the construction of electron density maps of both ribosomal subunits, showing recognizable morphologies and internal features similar to those observed by EM reconstructions of the corresponding ribosomal particle. The main features of these maps include elongated dense regions traceable as well separated RNA duplexes or single strands. Also seen are globular patches of lower density, readily distinguishable from the above, in which folds observed by NMR or crystallography in isolated ribosomal proteins at atomic resolution were detected. The intercomponents contacts identified so far reveal diverse modes of recognition. Metal clusters, attached at selected sites on the particles, are being exploited to facilitate unbiased map interpretation. In this way, two surface proteins were located and several surface RNA strands were targeted