Transcript Chapter 33 Slides

Biochemistry 2/e - Garrett & Grisham
Chapter 33
Protein Synthesis and
Degradation
to accompany
Biochemistry, 2/e
by
Reginald Garrett and Charles Grisham
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Biochemistry 2/e - Garrett & Grisham
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
• 33.7 Protein Degradation
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Biochemistry 2/e - Garrett & Grisham
Ribosome Structure and
Assembly
• E. coli ribosome is 25 nm diameter, 2520 kD in
mass, and consists of two unequal subunits that
dissociate at < 1mM Mg2+
• 30S subunit is 930 kD with 21 proteins and a 16S
rRNA
• 50S subunit is 1590 kD 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
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Ribosomal Proteins
• One of each per ribosome, except L7/L12
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
• Little known of structures - these proteins
are insoluble and difficult to study
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Ribosome Assembly/Structure
• If individual proteins and rRNAs are mixed,
functional ribosomes will assemble
• Gross structures of large and small
subunits are known - see Figure 33.3
• A tunnel runs through the large subunit
• Growing peptide chain is thought to thread
through the tunnel during protein synthesis
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Eukaryotic Ribosomes
• 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
• See Table 33.2 for properties
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Mechanics of Protein
Synthesis
• All protein synthesis involves three phases:
initiation, elongation, termination
• Initiation involves binding of mRNA and initiator
aminoacyl-tRNA to small subunit, followed by
binding of large subunit
• Elongation: synthesis of all peptide bonds - with
tRNAs bound to acceptor (A) and peptidyl (P)
sites. See Figure 33.5
• Termination occurs when "stop codon" reached
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Prokaryotic Initiation
• The initiator tRNA is one with a formylated
methionine: f-Met-tRNAfMet
• It is only used for initiation, and regular
Met-tRNAmMet 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
(see Figure 33.8)
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More Initiation
• 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 ribosomebinding site - is known as the ShineDalgarno sequence (see Figure 33.9)
• Initiation factor proteins, GTP, N-formyl-MettRNAfMet, mRNA and 30S ribosome form
the 30S initiation complex
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Events of Initiation
• 30S subunit with IF-1 and IF-3 binds
mRNA, IF-2, GTP and f-Met-tRNAfMet
(Figure 33.10)
• IF-2 delivers the initiator tRNA in a GTPdependent process
• Loss of the initiation factors leads to
binding of 50S subunit
• Note that the "acceptor site" is now poised
to accept an incoming aminoacyl-tRNA
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The Elongation Cycle
• The elongation factors are vital to cell function,
so they are present in significant quantities (EFTu 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-Ts recycles EF-Tu by exchanging GTP for
GDP
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Peptidyl Transferase
• This is the central reaction of protein synthesis
• 23S rRNA is the peptidyl transferase!
• The "reaction center" of 23S rRNA is shown in
Figure 33.14 - these bases are among the most
highly conserved in all of biology.
• Translocation of peptidyl-tRNA from the A site to
the P site follows (see Figures 33.12 & 33.15 and note that thenomenclature for Fig. 33.15 is
provided at the top of page 1103)
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The Role of GTP Hydrolysis
• Three GTPs are hydrolyzed for each
amino acid incorporated into peptide.
• Hydrolysis drives essential conformation
changes
• Total of five high-energy phosphate
bonds are expended per amino acid
residue added - three GTP here and
two in amino acid activation via
aminoacyl-tRNA synthesis
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Peptide Chain Termination
• 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
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Eukaryotic Protein Synthesis
See Figure 33.22 for the structure of the typical
mRNA transcript
• Note the 5'-methyl-GTP cap and the poly A tail
• Initiation of protein synthesis in eukaryotes
involves a family of at least 11 eukaryotic
initiation factors
• The initiator tRNA is a special one that carries
only Met and functions only in initiation - it is
called tRNAiMet but it is not formylated
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Eukaryotic Initiation
• Begins with formation of ternary complex of eIF-2,
GTP and Met-tRNAiMet
• This binds to 40S ribosomal subunit:eIF-3:eIF4C
complex to form the 40S preinitiation complex
• Note no mRNA yet, so no codon association with
Met-tRNAiMet
• mRNA then adds with several other factors, forming
the initiation complex (Fig. 33.23)
• Note that ATP is required!
• Proteins of the initiation complex apparently scan to
find the first AUG (start) codon
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Regulation of Initiation
Phosphorylation is the key, as usual
• At least two proteins involved in initiation
(Ribosomal protein S6 and eIF-4F) are
activated by phosphorylation
• But phosphorylation of eIF-2a causes it to
bind all available eIF-2B and sequesters it
• Note discussion of elongation and
termination on page 1112
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Inhibitors of Protein Synthesis
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Two important purposes to biochemists
These inhibitors (Figure 33.26) have helped unravel
the mechanism of protein synthesis
Those that affect prokaryotic but not eukaryotic
protein synthesis are effective antibiotics
Streptomycin - an aminoglycoside antibiotic - induces
mRNA misreading. Resulting mutant proteins slow the
rate of bacterial growth
Puromycin - binds at the A site of both prokaryotic and
eukaryotic ribosomes, accepting the peptide chain
from the P site, and terminating protein synthesis
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Diphtheria Toxin
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An NAD+-dependent ADP ribosylase
One target of this enzyme is EF-2
EF-2 has a diphthamide (see Figure 33.27)
Toxin-mediated ADP-ribosylation of EF-2
allows it to bind GTP but makes it inactive in
protein synthesis
One toxin molecule ADP-ribosylates many
EF-2s, so just a little is lethal!
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Ricin
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from Ricinus communis (castor bean)
One of the most deadly substances known
A glycoprotein that is a disulfide-linked
heterodimer of 30 kD subunits
The B subunit is a lectin (a class of proteins
that binds specifically to glycoproteins &
glycolipids)
Endocytosis followed by disulfide reduction
releases A subunit, which catalytically
inactivates the large subunit of ribosomes
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Ricin A subunit mechanism
• Ricin A chain specifically attacks a single,
highly conserved adenosine near position
4324 in eukaryotic 28S RNA
• N-glycosidase activity of A chain removes
the adenosine base
• Removal of this A (without cleaving the RNA
chain) inactivates the large subunit of the
ribosome
• One ricin molecules can inactivate 50,000
ribosomes, killing the eukaryotic cell!
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Protein Folding
• Proteins are assisted in folding by molecular
chaperones - called chaperonins
• Hsp60 and Hsp70 are two main classes
• Hsp70 recognizes exposed, unfolded regions
of new protein chains - especially hydrophobic
regions
• It binds to these regions, apparently protecting
them until productive folding reactions can
occur
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The GroES-GroEL Complex
• The principal chaperonin in E. coli
• GroEL forms two stacked 7-membered rings of
60 kD subunits; GroES is a dome on the top
• Nascent protein apparently binds reversibly
many times to the walls of the donut structure,
each time driven by ATP hydrolysis, eventually
adopting its folded structure, then being
released from the GroES-GroEL complex
• Rhodanese (as one example) requires
hydrolysis of 130 ATP to reach fully folded state
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Protein Translocation
An essential process for membrane proteins and
secretory proteins
• Such proteins are synthesized with a "leader
peptide", aka a "signal sequence" of about 1626 amino acids
• The signal sequence has a basic N-terminus, a
central domain of 7-13 hydrophobic residues,
and a nonhelical C-terminus
• The signal sequence directs the newly
synthesized protein to its proper destination
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Protein Translocation II
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Four common features
Proteins are made as preproteins containing
domains that act as sorting signals
Membranes involved in protein translocation
have specific receptors on their cytosolic faces
Translocases catalyze the movement of the
proteins across the membrane with metabolic
energy (ATP, GTP, ion gradients) essential
Preproteins bind to chaperones to stay loosely
folded
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Prokaryotic Protein Transport
All non-cytoplasmic proteins must be
translocated
• The leader peptide retards the folding of the
protein so that molecular chaperone proteins
can interact with it and direct its folding
• The leader peptide also provides recognition
signals for the translocation machinery
• A leader peptidase removes the leader
sequence when folding and targeting are
assured
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Eukaryotic Protein Sorting
Eukaryotic cells contain many membrane-bounded
compartments
• Most (but not all) targeting sequences are Nterminal, cleaveable presequences
• Charge distribution, polarity and secondary
structure of the signal sequence, rather than a
particular sequence, appears to target to
particular organelles and membranes
• Synthesis of secretory and membrane proteins
is coupled to translocation across ER membrane
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Events at the ER Membrane
• As the signal sequence emerges from the
ribosome, a signal recognition particle (SRP)
finds it and escorts it to the ER membrane
• There it docks with a docking protein or SRP
receptor - see Figure 33.31
• SRP dissociates in a GTP-dependent process
• Protein synthesis resumes and protein passes
into ER or into ER membrane; signal is
cleaved
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Protein Degradation
• Some protein degradation pathways are
nonspecific - randomly cleaved proteins
seem to be rapidly degraded
• However, there is also a selective, ATPdependent pathway for degradation - the
ubiquitin-mediated pathway
• Ubiquitin is a highly-conserved, 76 residue
(8.5 kD) protein found widely in eukaryotes
• Proteins are committed to degradation by
conjugation with ubiquitin
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Ubiquitin and Degradation
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Three proteins involved: E1, E2 and E3
E1 is the ubiquitin-activating enzyme - it forms a
thioester bond with C-terminal Gly of ubiquitin
Ubiquitin is then transferred to a Cys-thiol of E2,
the ubiquitin-carrier protein
Ligase (E3) selects proteins for degradation. the
E2-S~ubiquitin complex transfers ubiquitin to
these selected proteins
More than one ubiquitin may be attached to a
protein target
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