Lecture 14

Translation 3

Regulation of Translation
Protein Folding & Protein Degradation

Lodish 7th Ed. Chapter 8.4 p371-373 & 376-380

 Regulation of Translation (overview)
Advantages of regulation of gene expression at the translational level are the speed and
the readily reversible nature of the response to changes in physiological conditions
1. Global regulation of translation of most mRNAs
Pathways that control global protein synthesis in response to growth and stress
stimuli:
1.1 metazoan Target Of Rapamycin (mTOR) pathway - Stimulates translation
Rapamycin is an antibiotic produced by Streptomyces bacteria that inhibits general
protein synthesis (i.e., translation) and cell growth. The protein target inhibited by this
antibiotic is the protein kinase mTOR that phosphorylates multiple proteins involved
in ribosome biosynthesis and regulation of translation.
1.2 The eIF2 kinase pathways – Inhibit translation
Various stress-activated protein kinases phosphorylate eIF2 which in turn inhibits
eIF2B and leads to inhibition of translation initiation.

2. Regulation of translation of specific mRNAs

2.1 By sequence-specific RNA-binding proteins, which bind to particular
sequences or secondary structures in certain mRNAs to regulate their translation.
2.2 By small double-stranded RNAs called microRNAs (miRNAs), which inhibit
translation by hybridizing to specific mRNAs having partial nucleotide sequence
complementarity. See future lecture on RNA interference (RNAi).
1.1 The mTOR pathway controls protein synthesis & cell growth
Stress:
Growth factors Hypoxia
signaling Low energy
Low nutrients (amino acids)

mTOR kinase (Active) mTOR kinase (Inactive)

Part of mTORC1 complex (with GTP-bound Rheb) mTORC1 complex (GDP-bound Rheb)

Protein Ser/Thr Phosphorylation

4E-BP S6-Kinase Specific transcription factors for

RNA Pol I, Pol II, Pol III

Small rRNA Ribosomal tRNA

eIF4E ribosomal protein
S6 protein genes
(activated by
phosphorylation)
Translation
Initiation 4E-BP (eIF4E-Binding Protein)
Binds to eIF4E
(CAP-binding) inhibits eIF4E binding to CAP
When phosphorylated by mTOR cannot bind eIF4E
inhibition
Increased translation / protein synthesis activation
(Stimulates cell growth)
(See also Fig. 8-30 Lodish 7th Ed.)
 1.2 Stress-induced phosphorylation of eIF2 inhibits translation initiation

Amino acid ER stress dsRNA Heme

starvation (improper folding of (> 30 bp, viral RNAs) Deficiency (iron deficiency)
proteins in the ER)
(uncharged tRNAs)

activated
GCN2 PEK PKR HRI eIF2B is a GEF : Guanine nucleotide Exchange Factor

eIF2 kinases eIF2B-GTP

P
eIF2-GDP “Exchange”
does not occur

X
P
eIF2-GDP
eIF2B-GTP
GEF
eIF2-GDP eIF2-GDP eIF2-GTP

GDP
+ initiator tRNA-Met

Lodish 7th ed. Chapter 8.4, p378-379

Translation initiation
2.1 Regulation of specific mRNA translation by sequence-specific
RNA-binding proteins
Iron-dependent regulation of translation of ferritin mRNA
Precise regulation of cellular iron concentration is important as many cellular enzymes
contain Fe2+ as a cofactor (e.g. Krebs cycle enzymes) and free Fe2+ is toxic for cells.
Ferritin is an intracellular protein that binds and stores excess cellular iron.

IRE: Iron-Responsive Element

IRE-BP: IRE-Binding Protein

Bound to iron

sewer Binds iron

LN N

Fig.8-31 Lodish 7th Ed.

Chaperones and Protein Folding
•  Protein domains begin folding while the
protein is still being synthesized

Molten Globule
 Molten Globule
•  Open flexible protein structure present
immediately after protein synthesized
•  Secondary structure is formed
•  Most but not all of tertiary structure is formed.
 Within a few seconds of synthesis local
protein domains assume compact secondary
structures which are roughly in the correct
tertiary position

Molten Final
globule correct
form structure
Chaperones are required by most proteins to
fold into their native conformations

(Hsp70/Hsp40)

Partially folded protein

ATP
Correctly folded
Other Chaperones
ADP protein
(Hsp90, TriC)
(Hsp60 in mitochondria)
 How does the hsp 70
class of chaperones work?
•  Most proteins fold with hydrophobic amino acids
inside
•  When proteins are malfolded hydrophobic
amino acids are exposed to solvent
•  These chaperones bind the exposed
hydrophobic patches
•  Hydrolyze ATP massaging the hydrophobic
regions back into the protein and into the correct
conformation
During their first folding or with time
proteins can become misfolded

proteasome
 Proteasome
•  The proteasome is a large multisubunit enzyme/protease complex
•  The proteasome is the major path of degradation of proteins in eukaryotes
•  It has a 20S core that degrades the proteins
•  The entire 20S core consists of two rings each composed of 7 α and β subunits
•  The β subunits are the proteases (cleave proteins at hydrophobic, acidic & basic amino acids).
•  The core 20S proteasome is enclosed by two 195 kDa regulatory complexes (19S caps).
•  Ubiquitin-tagged proteins enter through one of two 19S regulators (which unfold the proteins).

19S
regulator cap
α
β 20S core
β
26S proteasome
α
19S
regulator cap

Lodish 7th ed. Chapter 3.4 p85-88

 The ubiquitin-proteasome
pathway for protein degradation

•  Multiple molecules of ubiquitin (a short protein of 76

amino acids) are conjugated to a lysine on the target
protein in a reaction involving three enzymes E1, E2
and E3.
•  Once ubiquitinated, more rounds of ubiquitination
take place (i.e. poly-ubiquitination).
•  The poly-ubiquitinated substrate is recognized by the
proteasome (by the 19 S regulator cap).
•  The proteasome rapidly unfolds the protein in an
ATP-dependent manner and hydrolyses the protein
to short peptides.
•  The ubiquitin molecules are released and used again.
•  Most of the released peptide products are then
hydrolyzed to amino acids by exopeptidases.
Lodish 7th ed. Chapter 3.4 p85-88
 Ubiquitination tagging
for protein degradation (in eukaryotes)

E2 = Ubiquitin-conjugating enzyme
Cysteine (several/many different)

E1 = Ubiquitin-activating enzyme
(only a few different types in a cell)

ubiquitin
poly E3 = Ubiquitin ligase
(very large variety, needed to
lag
Lodish 7th ed. Chapter 3.4 p85-88 recognize many substrates)
 Protein Degradation
•  Ubiquitin is bound to an Enzyme 1 (E1) cysteine and then transferred to
a cysteine of E2 and then to the targeted substrates via an E2-E3-
substrate complex
•  Ubiquitin is linked to the free ε-amino group on lysine in the target
protein
•  Each protein can be ubiquitinated at one or more lysine residues
•  The first ubiquitin added then becomes ubiquitinated and subsequent
ubiquitins are added to form a poly-ubiquitin chain on the protein
•  The ubiquitin chain is recognized by the 26S proteasome
•  Some proteins contain a single stable ubiquitin; these proteins are not
subject to degradation (e.g., histones are mono-ubiquitinated, see
future lecture)

Lodish 7th ed. Chapter 3.4 p85-88

 Protein Ubiquitination
Ubiquitin is conjugated via its C-terminus to the ε-NH2 side chain of lysine (K)
residues. The covalent bond created is called an isopeptide bond.

NH2
COOH

Lysine Protein
----------------NH-CH-CO-NH-CH-CO----------

(CH2)4 R
Ubiquitin 1
Ubiquitin 2 NH2----------------------CO-
K NH
NH2----------------------CO-
K NH
NH

Poly-ubiquitination of K residues (>4 ubiquitin molecules) --> protein degradation via the 26S
proteasome.

Oligo-ubiquitination (< 4 ubiquitin molecules) --> regulatory functions: ubiquitination of transcription

factors regulate their subcellular localization and activity…

Lodish 7th ed. Chapter 3.4 p85-88

 Ubiquitin-dependent protein degradation by the 26 S proteasome

ATP-dependent protein
unfolding and transfer
to the core

Lodish 7th Ed. Fig. 3-29

 Initiators of protein degradation

Removal of proteins that are toxic, inactive or damaged due to:

•  Misfolding
•  Oxidation
•  amino acid racemization (L to D)
•  Errors in translation (not well understood)
•  Early termination by stop codons when translation is out of
frame
•  Mutations in DNA/genes and hence in final proteins
•  Cell stress, such as heat stress

Removal of functional proteins that are not needed anymore due to:
•  Rapid change in cell status (e.g., regulatory stimuli, cell cycle)

Lodish 7th ed. Chapter 3.4 p85-88

Study Questions
•  Describe two general mechanisms for global regulation of mRNA translation into protein
•  Describe two mechanisms for regulation of translation of specific mRNAs (RNAi described later).
•  What is mTOR?
•  How dos eIF2B regulate mRNA translation?
•  By which mechanism does 4E-BP regulate mRNA translation?
•  What are "molten globules" and why do they arise during protein synthesis?
•  What are molecular chaperones? Name one class of them and tell what reactions they catalyze.
•  How does the hsp70 class of molecular chaperones work? What do they do?
•  What is ubiquitin?
•  What is the fate of poly-ubiquitinated proteins?
•  How does ubiquitin lead to protein degradation?
•  Describe the different types of enzymes involved in protein ubiquitination.
•  What is a proteasome?
•  What is the general structure of the proteasome 20S and 26 S assemblies?
•  Why are proteins degraded by cells?
•  What kind of linkage is an isopeptide bond?