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Chapter 33
Biochemistry 432/832
Protein Synthesis and
Degradation
December 05
Announcements
Protein Synthesis and Degradation
Central dogma of biochemistry:
DNA --> RNA --> protein
Transcription
Translation
Outline
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33.1 Ribosome Structure and Assembly
33.2 Mechanics of Protein Synthesis
33.3 Protein Synthesis in Eukaryotes
33.4 Inhibitors of Protein Synthesis
33.5 Protein Folding
33.6 Post-Translational Processing of Proteins
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33.7 Protein Degradation
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Proof that polypeptides (hemoglobin) grow by
addition of new amino acid residues to the
carboxyl end
Direction of chain growth
0 min
unlabeled
4 min
7 min
16 min
labeled
N-terminus
60 min
C-terminus
Dintzis experiment, 1961, rabbit reticulocytes labeled with leucine
Ribosomes
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Ribonucleoprotein particles
Found in the cytosol, mitochondria and
chloroplasts of all cells
Move along mRNAs and synthesize proteins
Bind and orient mRNAs and aminoacyl-tRNAs
Organize interactions between codons and
anticodons in aminoacyl-tRNAs
Catalyze the formation of peptide bonds between
adjacent amino acid residues
Ribosome Structure and
Assembly
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E. coli ribosome is 25 nm diameter, 2520 kDa in
mass, and consists of two unequal subunits that
dissociate at < 1mM Mg2+
30S subunit is 930 kDa with 21 proteins and a
16S rRNA
50S subunit is 1590 kDa with 31 proteins and two
rRNAs: 23S rRNA and 5S rRNA
These ribosomes and others are roughly 2/3 RNA
20,000 ribosomes in a cell, 20% of cell's mass
Organization of E.coli ribosomes
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Sedimentation
coefficient
 Mass (kDa)
 Major RNAs
 Minor RNAs
 RNA mass
 RNA proportion
 Proteins
 Protein mass
 Protein
Ribosome
Small
subunit
Large
subunit
70S
30S
50S
2520
930
1590
16S (1542 b) 23S (2904 b)
5S (120 b)
560
1104
60%
70%
21
31
370
487
40%
30%
1664
66%
857
34%
Ribosomal RNA operons in Escherichia coli.
Precursor RNA is cleaved to generate 23S, 16S
and 5S rRNA as well as tRNA
The 16S
rRNA fits
within the
30S
ribosomal
subunit
The rest of
space
(peripheral)
is occupied
by proteins
Ribosomal Proteins
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One of each per ribosome, except L7/L12 (same
proteins that differ at N-terminus) with 4
L7/L12 identical except for extent of acetylation
at N-terminus
Four L7/L12 plus L10 makes "L8"
Only one protein is common to large and small
subunits: S20 = L26
No similarity (Lys, Arg-rich). The largest is S1
(557 aa) , the smallest is L34 (46 aa)
Overall fold of proteins was established less
than a year ago
Ribosome Assembly/Structure
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If individual proteins and rRNAs are mixed,
functional ribosomes will assemble
 Structures of large and small subunits have
been determined in 2000/2001
 A tunnel runs through the large subunit
 Growing peptide chain is thought to thread
through the tunnel during protein synthesis
A 3D model for the E.coli ribosome
Two views
30S
70S
50S
Comparison of
ribosomes and
tRNAs (two
tRNAs may be
bound to a
ribosome)
E.coli ribosome
Image
reconstruction is
based on
cryoelectron
microscopy
Eukaryotic Ribosomes
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Mitochondrial and chloroplast ribosomes are
quite similar to prokaryotic ribosomes,
reflecting their supposed prokaryotic origin
 Cytoplasmic ribosomes are larger and more
complex, but many of the structural and
functional properties are similar
 Complexity and size of ribosomes are increased
from prokaryotes to lower eukaryotes to higher
eukaryotes
 Conservation of overall RNA structure as well
as specific segments of primary sequences
Properties of Eukaryotic Ribosomes
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Ribosome
Small
subunit
Large
subunit
Sedimentation
coefficient
Mass (kDa)
Major RNAs
Minor RNAs
80S
40S
60S
4220
RNA mass
RNA proportion
Proteins
Protein mass
Protein
2520
60%
1400
2820
18S (1874 b) 28S (4718 b)
5.8S (160 b)
5S (120 b)
700
1820
50%
65%
33
49
700
1000
50%
35%
1700
40%
Mechanics of Protein Synthesis
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All protein synthesis involves three phases:
initiation, elongation, termination
Initiation involves binding of mRNA and initiator
aminoacyl-tRNA to a small subunit, followed by
binding of a large subunit
Elongation: synthesis of all peptide bonds - with
tRNAs bound to acceptor (A) and peptidyl (P)
sites.
Termination occurs when "stop codon" reached
Two binding sites
Transfer of a polypeptide to the
amino group of amino acid
carried by the tRNA in the A site
Basic
steps
in
protein
synthesis
Aminoacyl tRNA is at the A
site; polypeptidyl-tRNA is
at the P site
One codon
translocation;
tRNA expulsion
Location of tRNA binding sites in a ribosome
Prokaryotic Initiation
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The initiator tRNA is one with a formylated
methionine: f-Met-tRNAfMet
It is only used for initiation, and regular MettRNAmMet is used instead for Met addition
N-formyl methionine is first aa of all E.coli
proteins, but this is cleaved in about half
A formyl transferase adds the formyl group
Structure of
N-formylmethionyltRNA[Met]
Differences
with other
tRNAs
More Initiation
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Correct registration of mRNA on ribosome
requires alignment of a pyrimidine-rich sequence
on 3'-end of 16S RNA with a purine-rich part of
5'-end of mRNA
The purine-rich segment - the ribosome-binding
site - is known as the Shine-Dalgarno sequence
Initiation factor proteins, GTP, N-formyl-MettRNAfMet, mRNA and 30S ribosome form the
30S initiation complex
Shine-Dalgarno sequences recognized by E.coli
ribosomes
Events of Initiation
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30S subunit with IF-1 and IF-3 binds mRNA,
IF-2, GTP and f-Met-tRNAfMet
 IF-2 delivers the initiator tRNA in a GTPdependent process
 Loss of the initiation factors leads to binding of
50S subunit
 The "acceptor site" is now poised to accept an
incoming aminoacyl-tRNA
Peptide chain
initiation
30S subunit (IF-3:IF1) binds mRNA
IF-2 delivers the
initiator f-Met-tRNA
to the P site
IF-2 dissociates
from 30S subunit
GTP hydrolysis is
accompanied by IFs
release and binding
of the 50S subunit
A structure of non-hydrolyzable analog of GTP
Allowed separation of GTP binding from GTP hydrolysis
The Elongation Cycle
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The elongation factors are vital to cell function, so
they are present in significant quantities (EF-Tu is
5% of total protein in E. coli )
EF-Tu binds aminoacyl-tRNA and GTP
Aminoacyl-tRNA binds to A site of ribosome as a
complex with 2EF-Tu and 2GTP
GTP is then hydrolyzed and EF-Tu:GDP
complexes dissociate
EF-T recycles EF-Tu by exchanging GTP for
GDP
Elongation factors
Factor
Mass
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EF-Tu
EF-Ts
43 kDa
74 kDa
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EF-G
77 kDa
Molecules/Cell Function
70,000 Binds tRNA-GTP
10,000 Displaces GDP
from EF-Tu
20,000 Binds GTP,
promotes
translocation of
ribosome
Peptide chain
elongation
Reaction of the tRNA-linked peptidyl chain with the
a-amino group of an adjacent aminoacyl-tRNA no energy is required for activation
Peptidyl Transferase
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This is the central reaction of protein synthesis
23S rRNA is the peptidyl transferase!
The "reaction center" of 23S rRNA - the catalytic
bases are among the most highly conserved in all
of biology.
Translocation of peptidyl-tRNA from the A site to
the P site follows
Ribosome is a
ribozyme
(catalytic rRNA)
Catalytic center
is located in the
50S particle
The peptidyl
transferase
center of 23S
rRNA
Ribosome is ribozyme
Puglisi JD, Blanchard SC, Green R, Nat Struct Biol 2000
Oct;7(10):855 Approaching translation at atomic
resolution.
Atomic resolution structures of 50S and 30S ribosomal particles
have recently been solved by X-ray diffraction
In the 50S structure, the active site for peptide bond formation
was localized and found to consist of RNA. The ribosome is thus
a ribozyme
In the 30S structure, tRNA binding sites were located
The 30S subunit particle has three globular domains, and relative
movements of these domains may be required for translocation of
the ribosome during protein synthesis
Ribosome structure
Yusupov MM, Yusupova GZ, Baucom A, Lieberman K, Earnest TN,
Cate JH, Noller HF.
Science 2001 Mar 29
Crystal Structure of the Ribosome at 5.5 A Resolution.
We describe the crystal structure of the complete Thermus thermophilus
70S ribosome containing bound mRNA and tRNAs at 5.5 A resolution.
All of the 16S, 23S and 5S rRNA chains, the A-, P- and E-site tRNAs,
and most of the ribosomal proteins can be fitted to the electron density
map. The core of the interface between the 30S small subunit and the
50S large subunit, where the tRNA substrates are bound, is dominated by
RNA, with proteins located mainly at the periphery, consistent with
ribosomal function being based on rRNA. In each of the three tRNA
binding sites, the ribosome contacts all of the major elements of tRNA,
providing an explanation for the conservation of tRNA structure. The
tRNAs are closely juxtaposed with the intersubunit bridges, in a way that
suggests coupling of the 20 to 50 A movements associated with tRNA
translocation with intersubunit movement.
The Role of GTP Hydrolysis
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Two GTPs are hydrolyzed for each amino
acid incorporated into peptide.
Hydrolysis drives essential conformation
changes
Total of four high-energy phosphate bonds
are expended per amino acid residue added
- three GTP here and two in amino acid
activation via aminoacyl-tRNA synthesis
Movement of tRNAs during translation
Relative positions of tRNA molecules in a ribosome
during peptidyl transfer and translocation
Peptide Chain Termination
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Proteins known as "release factors"
recognize the stop codon at the A site
Presence of release factors with a nonsense
codon at A site transforms the peptidyl
transferase into a hydrolase, which cleaves
the peptidyl chain from the tRNA carrier
Termination of
protein synthesis