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Subsections of About
1. Summary/Zusammenfassung
Subsections of 1. Summary/Zusammenfassung
Chapter 1
Summary đŹđ§
Ribosomal protein biosynthesis (translation) is a crucial process in all
domains of life. This work aims to investigate the process of
translation in the mammalian system by means of single particle
cryogenic electron microscopy (cryo-EM). The focus of this thesis lies
on the following two aspects of mammalian translation:
1) Translocation by the mammalian cytosolic 80S ribosome. Translocation
moves the tRNA2â˘mRNA module directionally through the ribosome during
the elongation phase of translation and is associated with large scale
conformational changes within both the ribosome and the bound tRNAs. It
is catalyzed by the GTPase eEF2 (EF-G in bacteria). Although knowledge
on translocation, especially in the bacterial system, has accumulated in
the past years, the detailed mechanisms are not fully understood. In
particular, the role of GTP hydrolysis is controversial and structural
knowledge on translocation in the mammalian system has been missing.
In this work, three high-resolution structures of in vitro
reconstituted authentic intermediates of translocation by the mammalian
80S ribosome are presented. They are trapped by the non-hydrolysable GTP
analog GMPPNP and contain, in contrast to similar experiments in the
bacterial system, the translocase eEF2 and a complete tRNAâ˘mRNA module.
Single-molecule imaging, carried out in collaboration with Prof. Scott
Blanchard and colleagues, revealed that GTP hydrolysis principally
facilitates rate-limiting, late steps of translocation, consistent with
the presented cryo-EM structures. Comparison with the bacterial system
showed that distinctions between bacterial and mammalian translocation
mechanisms originate from differential dissociation rates of deacylated
tRNA from the E site.
Further, a cryo-EM structure of a mammalian 80S ribosome containing a
complete tRNA2â˘mRNA module and eEF2â˘GDP is presented, which stems from a
sample prepared by in vitro translocating a PRE complex using
eEF2â˘GTP. In contrast to the GMPPNP-stalled translocation intermediates,
this structure gives insight into the interaction of unstalled eEF2 with
the 80S ribosome.
2) The influence of serum on the energy landscape of mammalian
translation and on the structure of ribosomal protein eS6. Serum
treatment of cells intervenes with many signaling pathways, but it is
not known if the energy landscape of translation is altered upon its
influence. Serum deprivation and restimulation can be used as a model
system to diminish and enhance phosphorylation of ribosomal protein eS6,
which is a eukaryote-specific protein on the small ribosomal subunit.
The phosphorylation of the C-terminus of eS6 has been investigated since
a long time, however, its mechanistic role has not been elucidated yet.
In particular, hardly anything is known on possible structural impacts
of eS6 phosphorylation.
The presented work reveals that serum deprivation and restimulation do
not have an impact on the energy landscape of translation for the ex
vivo derived cytosolic fraction of polysomes. However, the observation
of different yields of cell lysate from serum deprived and restimulated
cells led to the proposition of a new hypothesis that suggests cellular
redistribution of ribosomes. The phosphorylation of ribosomal protein
eS6, which strongly correlates with serum treatment, does not lead to
observable structural changes in the small ribosomal subunit.
Finally, the structural analysis and in silico sorting of the obtained
translation intermediates led to the identification of two previously
not observed substates of the 80S rotated PRE ribosome and to the
unprecedented visualization of two distinct, native inititation
complexes.
Chapter 2
Zusammenfassung đŠđŞ
Die ribosomale Proteinbiosynthese (Translation) ist ein zentraler
Prozess in allen Lebensdomänen. In der vorliegenden Arbeit wird der
Mechanismus der mammalischen Translation mithilfe der kryogenen
Elektronenmikroskopie (cryo-EM) untersucht. Der Fokus liegt hierbei auf
den folgenden zwei Aspekten der mammalischen Translation:
1. Die Translokation durch das mammalische, zytosolische 80S Ribosom.
Die Translokation ist die gerichtete Bewegung des tRNA2â˘mRNA Moduls
durch das Ribosom während der Elongationsphase der Proteinbiosynthese
und ist mit umfangreichen Konformationsänderungen des Ribosoms und der
gebundenen tRNAs assoziiert. Sie wird durch die GTPase eEF2 (EF-G in
Bakterien) katalysiert. Obwohl während der vergangenen Jahre viel ßber
die Translokation, vor allem im bakteriellen System, zusammengetragen
wurde, bleibt der genaue Mechanismus unverstanden. Insbesondere die
Rolle der GTP-Hydrolyse ist kontrovers und es fehlen strukturelle Daten
Ăźber die Translokation im mammalischen System.
In dieser Arbeit werden drei hochaufgelĂśste Strukturen in vitro
rekonstituierter, authentischer Translokationsintermediate des
mammalischen 80S Ribosoms präsentiert. Sie konnten mithilfe des
nicht-hydrolysierbaren GTP-Analogons GMPPNP eingefangen werden und
enthalten im Gegensatz zu ähnlichen Experimenten im bakteriellen System
die Translokase eEF2 und ein komplettes tRNA2â˘mRNA Modul. Die in
Kollaboration mit Herrn Prof. Scott Blanchard und seinen Kollegen
durchgefĂźhrte EinzelmolekĂźl-Bildgebung ergab, dass die GTP-Hydrolyse
hauptsächlich späte, geschwindigkeitslimitierende Schritte der
Translokation fĂśrdert, eine Beobachtung, die in Einklang mit den
präsentierten cryo-EM Strukturen steht. Der Vergleich mit dem
bakteriellen System schlieĂlich zeigt, dass Unterschiede zwischen
bakteriellen und mammalischen Translokationsmechanismen in verschiedenen
Dissoziationsraten der deacylierten tRNA von der E Stelle begrĂźndet
sind.
Desweiteren wird die cryo-EM Struktur eines mammalischen 80S Ribosoms
mit einem kompletten tRNA2â˘mRNA Modul und eEF2â˘GDP präsentiert, welche
aus einer Probe stammt, fßr deren Herstellung ribosomale Prä-Komplexe in
vitro mit eEF2â˘GTP transloziert wurden. Anders als die mit eEF2â˘GMPPNP
eingefangenen Translokationsintermediate gewährt diese Struktur Einblick
in die Interaktion von unmanipuliertem eEF2 mit dem 80S Ribosom.
2) Der Einfluss von Serum auf die Energielanschaft der mammalischen
Translation und auf die Struktur des ribosomalen Proteins eS6. Die
Behandlung von Zellen mit Serum greift in viele Signalwege ein, doch es
ist nicht bekannt, ob auch die Energielandschaft der Translation
beeinflusst wird. Serum-Deprivation und -Stimulation kann als
Modellsystem fĂźr die Verringerung und Steigerung der Phosphorylierung
des ribosomalen Proteins eS6, einem Eukaryoten-spezifischen Protein der
kleinen ribosomalen Untereinheit, angewendet werden. Die
Phosphorylierung des C-Terminus von eS6 wird seit langer Zeit erforscht,
jedoch ist ihre mechanistische Bedeutung bisher unbekannt. Vor allem
weiĂ man kaum etwas Ăźber mĂśgliche strukturelle Auswirkungen der
eS6-Phosphorylierung.
Die vorliegende Arbeit zeigt, dass Serum-Deprivation und -Stimulation
keinen Einfluss auf die Energielandschaft der Translation in der
zytosolischen Fraktion der ex vivo gewonnenen Polysomen hat. Die
Beobachtung eines Unterschieds in den Zelllysatausbeuten zwischen
Serum-deprivierten und -stimulierten Zellen fĂźhrte jedoch zu einer neuen
Hypothese, welche die zelluläre Umverteilung von Ribosomen nahelegt. Die
Phosphorylierung des ribosomalen Proteins eS6, welche stark mit der
Serumbehandlung korreliert, fĂźhrte zu keinen sichtbaren strukturellen
Veränderungen in der kleinen ribosomalen Untereinheit.
Zu guter Letzt fĂźhrte die Strukturanalyse und in silico Sortierung der
erhaltenen Translationsintermediate zu der Identifikation zweier bisher
nicht beobachteten Unterzustände des 80S rotierten Prä-Ribosoms sowie zu
der erstmaligen Visualizierung zweier verschiedener, nativer
Initiationskomplexe.
1. Summary/Zusammenfassung
Subsections of 2. Introduction
Common principles of protein biosynthesis
A current theory states that it was RNA that stood at the beginning of
life on our planet (Gesteland, R.F., Cech, T.R., 1999). Yet, the later
appearing proteins outperformed RNA in so many fields that those early,
self-sufficient RNA constructs are now extinct. Whereas RNA is composed
by a combination of basically four different types of nucleotides and is
relatively limited in its ability to form tertiary structure, proteins
are highly flexible chains built from twenty amino acids that can fold
in a larger variety of three-dimensional shapes. The amount of building
blocks (twenty compared to only four in RNA â leaving aside base
modifications) equips them with a high degree of adaptability to
different tasks and might be the reason for their superiority to RNA in
many fields.
To build such a peptide chain, amino acids have to be linked in the
correct order via peptide bond formation. Although there exist proteins
which are able to catalyze peptide bonds, like the sortase (Mazmanian et
al., 1999), in all kingdoms of life the responsibility for building
these peptide chains lies in a ribonucleoprotein particle: a
macromolecular machine called the ribosome.
General features of the ribosome
The ribosome consists of a large and a small subunit. Both are made up
of ribosomal RNA (rRNA) and ribosomal proteins
[(]{.FIGURE-HINWEIS}[Figure
1]{.FIGURE-HINWEIS}[)]{.FIGURE-HINWEIS}
[(]{.FIGURE-HINWEIS}[Animation
1]{.FIGURE-HINWEIS}[)]{.FIGURE-HINWEIS}. The basic mechanism
of protein synthesis is very similar in all domains of life: A messenger
RNA (mRNA) contains the sequence of the protein and is bound and read by
the small ribosomal subunit in collaboration with specific transfer RNAs
(tRNAs). tRNAs carry the amino acids and contain characteristic
anticodons, which establish base pairing with the respective codons on
the mRNA presented to them by the ribosome. Matching allows for addition
of the amino acid to the growing peptide chain. After all amino acids
have been added, the peptide chain is released from the ribosome and, if
needed, further processed by other cell components to become the final,
folded protein.
Although the three domains of life, bacteria, eukaryotes, and archaea,
all possess ribosomes for protein synthesis, the composition of the
ribosomes varies, leading to sometimes very different overall appearance
of ribosomes from different kingdoms of life (Amunts et al., 2015;
Behrmann et al., 2015; Dunkle et al., 2011; Melnikov et al., 2012;
Ramrath et al., 2018) ([Figure 2]{.FIGURE-HINWEIS}
).
Figure 1 blabla Image Caption
The eukaryotic cytoplasmic 80S ribosome is larger than the bacterial 70S
ribosome, containing additional RNA segments and additional proteins.
Comparison reveals that the eukaryotic ribosome possesses the same
conserved structures as the bacterial ribosome in its core ([Figure
2]{.FIGURE-HINWEIS}[A]{.FIGURE-HINWEIS}) and an outer shell
where eukaryote-specific elements are located ([Figure
1]{.FIGURE-HINWEIS}[, ]{.FIGURE-HINWEIS}[Figure
2]{.FIGURE-HINWEIS}) (Melnikov et al., 2012).
Not only the ribosomes themselves differ by certain features and are
differently sensitive to antibiotics (Yusupova and Yusupov, 2017), but
also the interacting factors that render translation possible are for
some stages of translation remarkably different (Andersen et al., 2006)
and might be an expression of the way the ribosome has been optimized
for its bacterial, archaean, or eukaryotic cell environment.
Aim 1
Aim 1
Anatomy
The size of the assembled 80S mammalian ribosome is about 4.3 MDa (Wool,
1979). Both subunits are made of ribosomal RNA (rRNA) and ribosomal
proteins (Wool, 1979). Upon joining of the large 60S (50S in bacteria)
subunit and the small 40S (30S in bacteria) subunit to the 80S ribosome
(70S in bacteria), a functionally important compartment is formed, the
so-called intersubunit space [(]{.FIGURE-HINWEIS}[Figure
1]{.FIGURE-HINWEIS}[), ]{.FIGURE-HINWEIS}which is one of the
main sites of action during protein synthesis. Across it span three
tRNA-binding sites, named A (Aminoacyl)-, P (Peptidyl)-, and E
(Exit)-sites. Additionally, the ribosome has specific factor binding
sites for the interaction with protein factors, like the P-stalk
[(]{.FIGURE-HINWEIS}[Figure
3]{.FIGURE-HINWEIS}[)]{.FIGURE-HINWEIS}.
[]{#rRNA is the catalytically active component of the ribosome}
rRNA is the catalytically active component of the ribosome
The rRNA possesses the catalytic activity to perform peptide bond
formation and is the main player in protein synthesis. The large (60S)
subunit contains three rRNA molecules: the 28S rRNA, the 5S rRNA and the
5.8S rRNA [(]{.FIGURE-HINWEIS}[Suplemental Figure
1]{.FIGURE-HINWEIS}[,
]{.FIGURE-HINWEIS}[Suplemental Figure
2]{.FIGURE-HINWEIS}). The small (40S) subunit
contains only one rRNA molecule, the 18S rRNA
[(]{.FIGURE-HINWEIS}[Supplemental Figure
3]{.FIGURE-HINWEIS}[)]{.FIGURE-HINWEIS}. The rRNA
regions responsible for mRNA-recognition, tRNA-binding and
peptidyl-transfer are highly conserved in all kingdoms of life
(Gesteland, R.F., Cech, T.R., 1999). Among these conserved regions are
the sarcin ricin loop (SRL) on the large subunit, interacting with
GTP-hydrolyzing protein factors that catalyze certain steps of
translation. In the peptidyltransferase center (PTC), also located on
the large subunit, the RNA alone is responsible for catalyzing the
formation of the peptide bond (Nissen et al., 2000; Spahn et al., 2000).
The small subunit 18S rRNA contains the decoding center (DC), which
monitors correct tRNA-anticodon matching to the mRNA codon.
Different from bacterial rRNA, eukaryotic 18S rRNA and 28S rRNA contain
several expansion segments, long elements of additional rRNA that are to
a great deal responsible for the big difference in size between
bacterial and mammalian ribosomes. The function of these expansion
segments is not yet clear, and their structural investigation is
hindered by their high flexibility and peripheral location, making it
very difficult to obtain high-resolution structural information (Ramesh
and Woolford, 2016; Yusupova and Yusupov, 2017). There is some evidence,
however, that expansion segments may play a role in ribosome biogenesis
(Ramesh and Woolford, 2016).
Despite the rRNAâs prominent role in translation, the ribosome would not
function without ribosomal proteins. They are important for rRNA folding
and assembly and stabilize the tertiary structure of rRNA and the
ribosomeâs functional centers. There are 33 ribosomal proteins on the
40S and 47 ribosomal proteins on the 60S subunit of the mammalian
ribosome. Following a recent convention (Ban et al., 2014) universally
conserved proteins are prefixed âuâ, unique bacterial ones âbâ, unique
eukaryotic ones âeâ, and unique archaeal ones âaâ
[(]{.FIGURE-HINWEIS}[Supplemental Figure
4]{.FIGURE-HINWEIS}[,
]{.FIGURE-HINWEIS}[Supplemental Figure
5]{.FIGURE-HINWEIS}[)]{.FIGURE-HINWEIS}.
[]{#The three-dimensional shape of the ribosome is optimized for its function}
The three-dimensional shape of the ribosome is optimized for its
function
The 40S subunit can be morphologically divided into several regions,
named after the 40S subunitâs resemblance in shape to a bird: Looking at
it from the solvent site, on top is the 40S âheadâ with its prominent
âbeakâ, below follows the âneckâ, and the âbodyâ is supplemented by the
âplatformâ, âshoulderâ and âfootâ domains ([Figure
3]{.FIGURE-HINWEIS}). In this work, a rough division into two
parts will be used: the 40S head (including beak) and the 40S
body/platform, comprising the remaining domains. Importantly, the link
between the 40S head and 40S body/platform is flexible and allows for
intrasubunit motions. Visible from the intersubunit space, there are the
three 40S tRNA binding sites that are distributed among the 40S head and
40S body/platform: A, P, and E ([Figure
3]{.FIGURE-HINWEIS}). The rRNA residues that constitute the
tRNA binding sites are well-conserved [(]{.FIGURE-HINWEIS}[Supplemental
Figure 1]{.FIGURE-HINWEIS}>, [Supplemental
Figure 2]{.FIGURE-HINWEIS}[,
]{.FIGURE-HINWEIS}[Supplemental Figure
3]{.FIGURE-HINWEIS}).
The large (60S) subunit is characterized by several landmarks; The
central protuberance, the P-stalk/stalk base and the L1 stalk ([Figure
3]{.FIGURE-HINWEIS}[C-D]{.FIGURE-HINWEIS}). From the solvent
side, one can see the ribosomal exit tunnel, from which the newly
synthesized protein emerges. The solvent side is to a large degree
covered by expansion segment ES7, the largest expansion segment of the
28S rRNA [Figure 3]{.FIGURE-HINWEIS}[C]{.FIGURE-HINWEIS}).
Looking on the 60S from the intersubunit space reveals the A-, P-, and
E-tRNA binding sites and the sarcin-ricin loop (SRL) ([Figure 3D]{.FH}).
Aim 1
Aim 1
Aim 1
1. Summary/Zusammenfassung
Subsections of 3. Aims
Aim 1
To visualize and mechanistically understand mammalian translocation.
Translocation is one of the least understood processes in protein
biosynthesis. Its correct completion is crucial for the continuation of
the translation elongation cycle, and ultimately for protein synthesis.
As upon translocation, the contacts between the ribosome and the
tRNA2â˘mRNA-module extensively rearrange, it is a process which requires
utmost accuracy. Efficient translocation depends on the action of the
specialized GTPase eEF2; however, detailed insights into the mechanism,
by which eEF2 catalyzes translocation is lacking and opposing hypotheses
are vividly discussed: The Brownian ratchet model, in which eEF2 is
supporting intrinsic conformational changes that lead to translocation,
and the power stroke model, according to which eEF2, being a motor
protein, actively moves the tRNAs in the direction of translocation
(Chen et al., 2016; Liu et al., 2014; Rodnina et al., 1997; Spirin,
2009).
Moreover, structural knowledge on tRNA translocation is dominated by
studies carried out in the bacterial system, and structural data on
mammalian tRNA translocation does not exist. Therefore, this work is
dedicated to studying how translocation is performed in the mammalian
system, using an in vitro reconstitution of a rabbit
80Sâ˘tRNA2â˘mRNAâ˘eEF2â˘GMPPNP complex. The goal is to obtain structures of
mammalian translocation intermediates to characterize mammalian
translocation and compare it to translocation in the bacterial and yeast
system.
Aim 2
To investigate the 80Sâ˘tRNA2â˘mRNAâ˘eEF2â˘GDP complex that is observed upon addition of eEF2â˘GTP to a programmed PRE complex.
To understand why eEF2 can stably bind to ribosomes and two tRNAs in
vitro (Budkevich et al., 2014), and how this fits to the model of
translocation, I look at an in vitro reconstituted rabbit
80Sâ˘tRNA2â˘mRNAâ˘eEF2â˘GDP complex.
Aim 3
To revisit the mammalian polysome landscape to find out if serum deprivation influences the distribution of states.
Cryo-EM of actively translating polysomes gives insight into the energy
landscape of mammalian translation (Behrmann et al., 2015). Among other
stimuli, serum deprivation and subsequent serum restimulation has been
hypothesized to influence translation, e.g. via changing polysome
patterns (Duncan and McConkey, 1982; Viero et al., 2015). I want to
investigate if overnight serum deprivation and 30 minutes of serum
restimulation can change the energy landscape of translation.
Aim 4
To look at the phosphorylation site of ribosomal protein eS6 and its possible impact on its surrounding structures to find out the role of eS6 phosphorylation.
The investigation of structural changes induced by eS6 phosphorylation
is the fourth aim of this thesis. eS6 is a eukaryote-specific protein of
the 40S subunit. It undergoes phosphorylation in response to various
stimuli, including serum deprivation/restimulation. Surprisingly, there
are almost no works tackling the role of eS6 phosphorylation from a
structural perspective. Since structural information on the eukaryotic
ribosome from crystal structures and cryo-EM density maps have emerged
in the last years, eS6 could be structurally characterized in yeast and recently also in mammalian
ribosomes. However, the last residues of the C-terminus of eS6,
including all five phosphorylatable serines, are missing in the
available structures, and similarly the neighbouring expansion segments
are not well resolved, such that the phosphorylation and its structural
consequences are not characterized yet (Behrmann et al., 2015; Ben-Shem
et al., 2011). This thesis aims to investigate the C-terminal region of
eS6 and its surrounding.
