PROTEOSTASIS

2A

Translation:
mRNA-directed Biosynthesis
of Proteins

How are proteins synthesized, folded, and

cotranslationally modified in the secretory pathway ?
1
 Translation
Activation of Amino Acids: Aminoacyl-Adenylation 2A
Hoagland & Zamecnik
discovered that amino
acids were activated
Amino Acid when incubated whith
ATP and the cytosolic

ATP fraction of liver

extracts A
General Concepts: Amino acids become
attached to a heat-
5´-Aminoacyl Adenylate
Energy for specificity of amino stable soluble RNA,
acid recognition, bond discoverd by Robert
Holley and later
formation and energy storage.
termed tRNA,
representing the
Mixed anhydride bond Conduction of an ordered, adaptor between
process requires energy
(the adenylate) is highly mRNA and amino
acids hypothezed by
energetized and can be Energy to overcome the Crick B
nucleophiolically attacked tendency to reach a maximum
of entropy The enzymatic activity
of liver extracts
catalizing this
important reaction is
due to so-called
Aminoacyl-tRNA
Amino acid transfered to Synthethases C
tRNA & linked via an Two step reaction is performed by
Ester-Bond Amino Acyl tRNA Synthetases 2
 Translational Initiation (1)
Pre-Initiation Complexes
Eukaryotic mRNA 5´-Cap: methylated Guanin-Nucleotide
linked via 5´5´-bond to the 5´-end 2B
 eIF1/3/5 prevent 60S binding
 eIF4E: Cap Binding
60S
 eIF1A occupies A-site RNA Helicase  eIF4G: eIF4E Binding

eIF2 – GTP
Helicase Activator
 eIF4A/B (helicase) driven
scanning requires ATP
eIF4F-Complex

 Structurless 5´end – a
The GTP-bound initiator tRNA prerequisite for 40S binding &
43S
binding factor triggers
AUG scanning mechanism
association of initator tRNA with
the P site in the absence of
mRNA  Scanning for AUG
 GTP Hydrolysis occures
after AUG recognition
48S  Prerequisite for 60S binding

Watson, J. D., Baker, T.A., Bell, S.P., Gann, A., Levine, M., Losick, R. (2011). Alberts, B. et al. (2015). MBO of the Cell, 6th Edition
Molekularbiologie, 6. Auflage PEARSON Studium Kap. 14 „Translation“, S. 504-566 Garland Science: Chapter 6: From RNA to Protein 3
Translational Initiation (2)
2B
48S Pre-Initiation Complex
Scanning for AUG

Principle:
Principle:
Cascades of Protein-Protein Interaction for
Energy Consumption for conformational changes and
organizing stepwise conduction of
altered protein connectivity and function
processes
 eIF2 – GTP: After successful scanning &
binding of initiator tRNA to AUG eIF5 eIF2 – GDP
stimulates eIF2 to hydrolyse GTP  Consequence of GTP hydrolysis is
the release of eIF2-GDP
eIF5B – GTP  Hydrolysis enables release of:
 binding of a second initiator tRNA eIF3, eIF1, eIF5, eIF4B
binding protein, eIF5B-GTP is now
allowed to occur
 eIF5B-GTP binds initiator tRNA &
stimulates 60S association

Dissociation
 eIF5B-GDP & A
Binding of 60S site occupier
triggers eIF5B-GTP 80S eIF1A, dissociate
hydrolysis

80S Initiation Complex 4

Translation: Elongation
EF-Tu: Aminoacyl-tRNA „Escort Service“ to the A Site Prokaryotes & Eukarya 2B
1 Elongation Factor eEF-Tu

 Only eF-Tu-GTP binds 3´end of tRNA , masks the

aminoacyl residue and prevents premature
condensation

 prevents premature hydrolysis of aminoacyl tRNA

 eF-Tu reveals low intrinsic GTPase Activity

 hydrolysis of GTP : tRNA-de-loading of EF-Tu

 60S factor binding site can stimulate hydrolysis

(GTPase activity) if correct codon-anticodon base

pairing allows EF-Tu to fit into the factor binding site,

Handouts, Scripts & PDF material only for internal usage ! 5

Translation: Elongation
Role of EF-Ts What happens with eEF-Tu-GDP ? Reloaded !!!
Prokarya & Eukarya
2B
1 Elongation Factor eEF-Ts

 EF-Ts stimulates the dissociation of GDP

from EF-Tu-GDP

 EF-Tu-GDP has a low dissociation rate, since

EF-Tu without nucleotide is structurally
instable

 EF-Ts is a Guanine nucleotide Exchange

Factor (GEF) specific for EF-Tu

 EF-Ts dissociates if GTP has bond the EF-tu

Handouts, Scripts & PDF material only for internal usage !

6
Translation: Elongation
Peptidyltransfer Reaction: Unraveling the Mechanism by structural Analysis 2B
 Ribosome encreases catalysis 10-million-(107-) fold as compared to free aminoacyl-tRNAs in solution

2  Ribosomes: no amino acid side chains in 1.8 nm distance of the peptidyl transfer active site
 N-term. of L27 protein (E. coli) in close distance; 9aa-deletion reduces rate only to 30-50%;

Peptidyltransfer Center

P A

50S
Subunits
rRNA

 general base catalysis: abstraction of a proton from the attacking amino group
 general acid catalysis donation of this proton to the tetrahedral intermediate
7
Translation: Elongation
Contributions of Peptidyl-tRNA and 23S rRNA in the Catalytic Mechansim 2B
 Role of Peptidyl-tRNA in P Site: Substrate-assistence during catalysis
 Elimination of the 2´-Hydroxyl Group of Ribose reduces velocity of peptidyltransfer 106-fold

Acylated
Aminoacyl-
tRNA in A site

Proton rRNA involved in

Shuttling Entropic Catalysis:
Basepairing of 23S
rRNA with CCA-tail
of tRNAs in A-and P
sites leads to
Deacylated positioning of
Peptidyl-tRNA a-Amino group of
in P site Aminoacyl-tRNA
and Carbonylgroup
of Peptidyl-tRNA

1. Conclusion:The Ribosome is a Ribozyme

 Model: 2´-OH donates Proton to
neighbouring 3´-oxygen of the RNA not Protein is the ribosomal catalyst
ester linkage and takes a proton 2. Translocation of the Ribosome is required
from the attacking a-amino group; Handouts, Scripts & PDF material only for internal usage ! 8
 Translation: Elongation Prokarya
The Mechanism of Translocation & Eukarya 2B
EF-G fits into factor binding centre of 60S & acts as molecular wedge

GDP-
 After transpeptidylation the
associated EF-
peptide chain is linked to the G is released
tRNA in the A site from the A
site due to an
 GTP-associated EF-G binds the altered
factor binding centre close to the confirmation

A site

Deacylated
 EF-G drives translocation of the tRNA leaves
ribosome relative to the mRNA in the ribosome
at the E site
5´-3´direction

 Hydrolysis of GTP is

3 accompanied by conformational
change Handouts, Scripts & PDF material only for internal usage ! 9
Translation: Termination
Release Factors Prokarya & Eukarya
2B
 Class-I-release
factors: RF1 binds at A
site if a Stop Codon
(UAA, or UAG) is
present*
 triggers peptide
release through GGQ
motif within the
peptidyl transferase
center

 Class-II-factors bind after

peptide release and drive
dissociation of class-I-factors
 Release factor 3 (RF3, a G After GTP hydrolysis, RF3 affinity
protein) binds at A site in its for the factor binding center
GDP-bound state drops dramatically
RF-3-GDP is released
 GDP/GTP exchange induces a
conformational change in RF3
which „kickes off“ the RF1 *RF2 recognises UGA or UAA
Handouts, Scripts & PDF material only for internal usage ! 10
PROTEOSTASIS
Regulation of Translation 2C
Initiation of translation is regulated at the level of assembly of the eIF4F complex, which circulizes the mRNA

Majorly, eukaryotic mRNAs are bound to the ribosome as a

complex with a number of specific binding proteins (eIF4F
complex). Several of these tie together the 5´ and 3´ ends
of the message.
The eIF3 factor takes responsibility to bridge the 40S
subcomplex and eIF4F.

Alternatively, Cap-independent initation mechanisms are

based on internal ribosomal entry sites (IRES) which are
found on viral as well as on cellular mRNAs

Signaling pathways such as those via mTOR

Kinase target 4E-Binding Proteins, which normally
compete the eIF4E-eIF4G interaction, necessary for
eIF4F-dependent translational initiation

Unphosphorylated 4E-BPs are translation inhibitors, due to their high affinity to eIF4E.
4EBP-4E interactions prevent 4G binding and eIF4F assemby. 11
PROTEOSTASIS
Regulation of initiation by
Regulation of Translation
2C
Growth factors are activating
eIF4E-Binding Proteins (4E-BPs) mTOR kinase, which
phosphorylates 4E-BP
Proteinkinase mTOR phosphorylates
the 4E-BP proteins 4E binding pocket.
Via inhibition of the important 4E-BP
task active mTOR kinase increases
cellular protein biosynthesis Unphosphorylated 4E-BPs
compete with eIF4G for eIF4E
binding. Phosphorylated 4E-
BPs do not bind to eIF4E and
are unable to compete with the
eIF4G protein

Viruses can block global

translation, but profit from
alternative initiation pathays
shown on the next slide ….
Sonenberg, N. and Hinnebusch, A.G. (2009).
Regulation of Translation Initiation
in Eukaryotes: Mechanisms and Biological
Targets. Cell 136: 731–745

12
PROTEOSTASIS
Internal Ribosomal Entry Site (IRES)
IRES-dependent Regulation of Translation
2C
 Expression of genes bearing IRES
elements in their mRNAs is controlled
“Translation of cellular mRNAs via by multiple molecular mechanisms
initiation at internal ribosome
entry sites (IRESs) has received  with IRES-mediated translation favored
increased attention during recent under conditions when cap-dependent
years due to its emerging translation is compromised
significance for many physiological
and pathological stress conditions  Some viruses create advantageous
in eukaryotic cells. “ scenarios for translating their own
mRNAs in favor of cellular mRNAs
 competitive action of IRES
transacting factors (ITAFs) plays Martinez-Salas E, Francisco-Velilla R, Fernandez-
a pivotal role in IRES-mediated Chamorro J and Embarek AM (2018) Insights
into Structural and Mechanistic Features of Viral
translation and thereby IRES Elements. Front. Microbiol. 8:2629. doi:
controls cell-fate decisions 10.3389/fmicb.2017.02629
leading to either pro-survival
stress adaptation or cell death.

Komar & Hatzoglou (2011)

Cell Cycle 10:229-240
13
2-4
PROTEOSTASIS Summary: Lecture 2
Roles of Translational Initiation Factors
Regulation of Translation
Eukaryotic cells have At the 3´end, the mRNA is bound by the
at least nine initiation factors. poly(A) binding protein (PAB; not shown in this
Excercises in picture)
Bioanalytics:
A complex called eIF4F, which includes the
Describe and combine proteins eIF4E, eIF4G, and eIF4A, binds to the
experimental techniques 5 cap through eIF4E.
demonstrating the physical
protein-protein interaction Global translation can be switched off by
of individual proteins such inhibiting the cap-dependent formation of
eIF4F - Complex eIF4F.
as eIFs E and G or the
composition of protein
complexes of higher In eIF4F, the protein eIF4G binds to both eIF4E
order. 43S Preinitiation
and PAB, effectively tying them together.
Complex
The recruited protein eIF4A has an RNA
helicase activity, which is activated by the eIFB
protein. The helicase removes secondary
mRNA structure to allow scanning of the
ribosome for a start codon.

It
48Sis the eIF4F complex that associates with the
14 40S ribosomal subcomplex
48S - Complex