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Strukturelle Studien am 70S Ribosom von Escherichia coli
No full textTitelblatt
Inhaltsverzeichnis
Allgemeine Einleitung
Kapitel I Einleitung
Kapitel I Ergebnisse und Diskussion
Kapitel I Material und Methoden
Kapitel I Abbildungen und Tabellen
Kapitel II Einleitung
Kapitel II Ergebnisse und Diskussion
Kapitel II Material und Methoden
Kapitel II Abbildungen und Tabellen
ReferenzenThe ribosome is a large ribonucleo-protein complex that is required for
protein biosynthesis in all kingdoms of life. In this thesis, functional
states of the complete Escherichia coli (E. coli) 70S ribosome have been
studied using a combination of x-ray crystallography and biochemical
approaches. One part of this thesis concerns the regulation of ribosome
function under cellular stress, such as cold shock. Together with Antón Vila-
Sanjurjo, a former postdoctoral fellow in the laboratory of J.H.D. Cate, I was
able to obtain a 11Å crystal structure of the complete 70S E. coli ribosome
bound to the stress response protein Protein Y (Vila-Sanjurjo, Schuwirth et
al. 2004). The structure was complemented by RNA - footprinting and binding
data, which enabled us to deduce a mechanism for ribosome inactivation during
cold shock. A decrease in temperature had been found to result in the
induction of Protein Y expression, which binds and stabilizes the 70S
ribosome, thereby serving as a storage protein for the translation machinery.
Here, I found that PY binds mainly to the ribosomal P site where it is able to
compete with Initiation Factor (IF) 1 and IF3 thereby blocking translation
initiation in the cold. Once normal growth conditions recur (37°C), the
initiation factors are able to overcome the PY inhibition of protein synthesis
allowing ribosomes to follow the pathway into active translation. Moreover, I
obtained a new crystal form of the E. coli ribosome that diffracts up to
atomic resolution. I was able to solve the structure of the 70S ribosome to
3.5 Å resolution, which allows us for the first time to obtain a structural
view of the entire ribosome at near atomic resolution. Two ribosomes within
the asymmetric unit of the crystals were found to have two distinct
conformations, which are hypothesized to reflect two functional stages of the
ribosome within the elongation cycle. Additionally, I was able to soak the
antibiotic kasugamycin (ksg) as well as the Ribosome Recycling Factor (RRF)
into the new crystal form, which resulted in difference maps of up to 3.5 Å
resolution for the ksg-bound ribosome and 4.5 Å resolution for the RRF-bound
ribosome. These ribosomal complexes help to explain how these factors may
influence ribosomal activity. The 3.5 Å structure of the empty 70S E. coli
ribosome, and the observation that the new crystal form is suitable for
binding experiments involving small chemical ligands as well as larger protein
factors, raises our capabilities of obtaining atomic resolution structures of
ribosomal complexes in different functional states. Combined, such structures
in the future should allow for the recapitulation of the complete
translational cycle at the ribosome in great molecular detail. The structural
analyses presented in this thesis provide a basis for attempts in the near
future to obtain further high-resolution structures of ribosomal complexes
trapped at different stages of the translational cycle, with the ultimate goal
of unraveling the molecular mechanisms involved in protein biosynthesis.Die Funktion des Ribosoms, ein Protein Ribonucleinsaeure Komplex,
beschraenkt sich auf die Proteinbiosynthese. Im Rahmen dieser Doktorarbeit
wurden kristallographische und biochemische Experimente dazu verwendet, um
strukturelle Prozesse innerhalb des Ribosoms von Escherichia coli (E. coli)
aufzudecken. Einer der Regulationsmechanismen des Ribosoms waehrend kaelte-
schock Bedingungen wurde aufgeklaert (Vila-Sanjurjo, Schuwirth et al. 2004).
Eine 11 Å Struktur des Ribosoms im Komplex mit Protein Y, einem Kaelteschock-
Protein in E. coli, konnte zusammen mit Antón Vila-Sanjurjo, einem ehemaligen
Mitarbeiter aus dem Labor von J. H. D. Cate, geloest werden. Neben der
Struktur halfen biochemische Experimente (RNS - foot printing und
Bindungstudien), einen Mechanismus fuer die Inhibition des Ribosoms waehrend
Kaelteschock zu postulieren. Ein Herabsinken der Temperatur fuehrt zur
Expression von Protein Y, welches and das Ribosom bindet, um es zu
stabilisieren. Hier konnte ich zeigen, dass Protein Y den Initiations-Schritt
der Translation in der Kaelte inhibiert, indem es mit IF1 und IF3
kompetitiert. Sobald die normalen Temperaturbedingungen wieder hergestellt
sind, wird Protein Y durch die Initiationsfaktoren verdraengt, die den Prozess
der Translation iniziieren. Darueber hinaus war es mir moeglich, eine neue
Kristallform fuer das E. coli 70S Ribosom zu finden, welche Roentgenstrahlen
bis zu einer atomaren Aufloesung von 3Å streut. Zum ersten mal wurde die
Struktur des gesamten 70S Ribosoms bis zu einer Aufloesung von 3.5 Å geloest.
Diese ermoeglicht es uns einen atomaren Einblick in das Ribosom zu erlangen.
Die asymmetrische Einheit der Kristalle beinhaltet zwei 70S Ribosomen-
Molekuele, die in zwei verschiedenen Konformationen vorherrschen. Diese
koennten das Ribosom in zwei unterschiedlichen Staaten des Translations-
Zyklusses widerspiegeln. Zudem war es mir moeglich, das Antibiotikum
kasugamcyin (ksg) sowie den Ribosome Recycling Factor (RRF) in die Kristalle
diffundieren zu lassen. Die 70S/ksg Kristalle streuten Roentgenstrahlen bis
3.5 Å, die 70S/RRF Kristalle bis 4.5 Å. Die Bindungsstelle der beiden Liganden
im Ribosom konnte so gezeigt werden. Die 3.5 Å Struktur des 70S Ribosoms sowie
die Difussionsversuche mit unterschiedlichen Liganden erhoeht die
Wahrscheinlichkeit, dass hoffentlich in naher Zukunft, hochaufloesende
Kristallstrukturen von funktionellen Ribosomenkomplexen geloest werden
koennen. Dies wuerde unser Wissen ueber den Mechanismus der Proteinbiosynthese
in grossem Masse erweitern. Die hier gezeigten strukturellen Analysen gelten
als Voraussetzung fuer weitere hochaufloesende Strukturen des Ribosoms und
seiner Ligande, die uns, hoffentlich in naher Zukunft, einen atomaren Einblick
in den Mechanismus der Proteinbiosynthese gewaehren
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
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koamabayili/VECTRON-author-checklist: VECTRON author checklist
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Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
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