CHAPTER 27

Protein Metabolism

Learning goals:
• The genetic code
• Synthesis of aminoacyl-tRNA
• Translation: RNA-dependent protein synthesis
• Protein targeting and degradation

1
 Content

27.1 The genetic code

27.2 Protein Synthesis

27.3 Protein Targeting and Degradation

2
 Regulation of Protein Synthesis

• Protein synthesis is highly energy demanding,

so it is highly coordinated.
• Protein synthesis can use 90% of the chemical
energy of a cell.
• Number of copies of protein produced
corresponds to number of copies needed.
• Proteins are targeted to cellular locations.
• Degradation keeps pace with synthesis.

3
 Protein Synthesis Is a
Very Complex Process

• In eukaryotes, requires >300 biomolecules:

– >70 ribosomal proteins
– ~20 aa activation enzymes
– ~20 protein factors for initiation, elongation,
and termination of peptides
– ~100 additional enzymes for final processing
– ~40 kinds of tRNAs and rRNAs

4
 Three Early Advances in
Understanding Protein Synthesis

1. Proteins synthesized at ribosomes

2. Amino acids activated for synthesis by attachment to
tRNA via aminoacyl-tRNA synthetases
3. tRNA acts as an “adapter” to translate mRNA into
protein

5
Rough Endoplasmic Reticulum

6
 Early History of Protein Synthesis
Research
• 1955: Paul Zamenick’s group identifies ribosome as the site of
protein synthesis.
• 1956: Volkin and Astrachan find RNA that is like DNA but do
not realize the importance of this finding.
• 1958: Zamenick and Hoagland find that amino acids became
attached to what is now known as tRNA.
• 1960: Sydney Brenner realizes at a Good Friday meeting with
Crick and Francois Jacob that the DNA-like RNA of Volkin and
Astrachan is the messenger RNA.

7
 The Genetic Code for Proteins
Consists of Triplets of Nucleotides
• There are 20 common, genetically encoded amino
acids.
• A four-letter code in groups of two is insufficient (16).
• A four-letter code in groups of three IS sufficient (64).
• Living organisms use nonoverlapping mRNA code with
no punctuation.

8
Adapter (tRNA) Brings Amino Acid to mRNA

9
 Overlapping versus
Nonoveralapping Code

10
 Breaking the Genetic Code

• One of the greatest scientific developments of the

twentieth century
• First clue from Nirenberg and Matthaei:
– poly(U) and 20 radiolabeled amino acids fed to E.coli
only *Phe produced
• Hence, UUU codes for Phe.

• Another step forward from Khorana:

– used defined mRNAs in planned patterns
• that is, (AC)n (alternating ACA and CAC codons  His and Thr)

11
 Features of the Genetic Code

• The code is written in the 5’  3’ direction.

• Third base is less important in binding to tRNA.
• The first codon establishes the reading frame.
– If reading frame is thrown off by a base or two, all
subsequent codons are out of order
• 61/64 codons code for amino acids.
• There are three are termination codons:
– UAA, UGA, UAG
• AUG = initiation codon (as well as Met codon)
12
Nucleotide Code Dictionary

13
 Most Amino Acids Have More Than
One Codon

• Some codons are less subject to causing a mutation

in an amino acid sequence because of degeneracy
or because of the abundance of such tRNAs.
• There are 20 amino acids with 61 possible codons.
• Only Met and Trp have a single codon.

14
Codon Correlation with Amino Acids

TABLE 27-3 Degeneracy of the Genetic Code

Number of Number of
Amino acid Amino acid
codons codons
Met 1 Tyr 2
Trp 1 Ile 3
Asn 2 Ala 4
Asp 2 Gly 4
Cys 2 Pro 4
Gln 2 Thr 4
Glu 2 Val 4
His 2 Arg 6
Lys 2 Leu 6
Phe 2 Ser 6

15
 The Genetic Code Is Universal,
With a Few Exceptions
• It is used by prokaryotes and eukaryotes, across
species.
• Mitochondria contain DNA and use a slightly
different code.
– UGA encodes Trp in vertebrate mtDNA (instead of STOP).
– AGA/AGG encodes STOP in vertebrate mtNDA (instead of
Arg).
• Mitochondria encode their own tRNAs, using 22
instead of 32.

16
 “Wobble” Pairings in tRNA with mRNA
Can Occur in the Third Base

• The third base of a codon can form noncanonical base

pairs with its complement (anticodon) in tRNA.
• Some tRNAs contain Inosinate (I), which can H-bond
with U,C, and A.
– These H-bonds are weaker and were named by Crick as
“wobble” base pairs.
– Example: In yeast, CGA, GCU, and CGC all bind to tRNAArg,
which has the anticodon 3’-GCI-5’.
• Although sequences are usually written 5’3’, the anticodon here
is written 3’5’ to illustrate its bonding to the mRNA codons.

17
 Molecular Recognition of Codons in
mRNA by tRNA
• The codon sequence is
complementary with the
anticodon sequence.
• The codon in mRNA base
pairs with the anticodon
in mRNA via hydrogen
bonding.
• The alignment of two
RNA segments is
antiparallel.
18
19
 The Genetic Code Is Resistant
to Mutations
• Degenerate code allows certain mutations to
still code for the same amino acid.
– “silent” mutations―different nucleotide in DNA
but same amino acid in protein
• Mutation in the first base of a codon usually
produces a conservative substitution.
– Example: GUU  Val but AUU Leu

20
 Some mRNAs Are Edited Before
Protein Synthesis
• Editing involves alteration, addition, or deletion of nucleotides
in mRNA.
• Editing uses guide RNAs (gRNAs) that temporarily hybridize
with the mRNA and act as templates for editing.

21
 Deamination Reactions Yield

• RNA editing by
alteration of
nucleotides most
commonly involves
the enzymatic
deamination of
adenosine or
cytidine residues.

22
 Five Stages of Protein Synthesis
1. Activation of amino acids
– tRNA aminoacylated
2. Initiation of translation
– mRNA and aminoacylated tRNA bind to ribosome
3. Elongation
– cycles of aminoacyl-tRNA binding and peptide bond
formation…until a STOP codon is reached
4. Termination and ribosome recycling
– mRNA and protein dissociate, ribosome recycled
5. Folding and posttranslational processing
– catalyzed by a variety of enzymes
23
Overview of Protein Synthesis

24
 Components Required for the Five Major Stages of Protein
TABLE 27-5
Synthesis in E. Coli
Stage Essential components
1. Activation of amino acids 20 amino acids
20 aminoacyl-tRNA syntheses
32 or more tRNAs
ATP
Mg2+
2. Initiation mRNA
N-Formylmethionyl-tRNAfMet
Initiation codon in mRNA (AUG)
30S ribosomal subunit
50S ribosomal subunit
Initiation factors (IF1, IF2, IF3)
GTP
Mg2+
3. Elongation Functional 70S ribosomes (initiation complex)
Aminoacyl-tRNAs specified by codons
Elongation factors (EF-Tu, EF-Ts, EF-G)
GTP
Mg2+
4. Termination and ribosome Termination codon in mRNA
recycling Release factors (RF1, RF2, RF3, RRF)
EF-G
IF3
5. Folding and posttranslational Chaperones and folding enzymes (PPI, PDI); specific enzymes, cofactors, and
processing other components for removal of initiating residues and signal sequences,
additional proteolytic processing, modification of terminal residues, and attachment
of acetyl, phosporyl, methyl, carboxyl, carbohydrate, or prosthetic groups
25
 The Ribosome Is a Key Player
in Protein Synthesis
• Make up ~25% of dry weight of bacteria
• ~65% rRNA, 35% protein
– rRNA forms the core.
– RNA does the catalysis of peptide bond formation.
• Made of two subunits bound together (30S and 50S) in
bacteria, with mRNA running through them

TABLE 27-6 RNA and Protein Components of the E. Coli Ribosome

Number of different Total number of
Subunit proteins proteins Protein designations Number and type of rRNAs
30S 21 21 S1–S21 1 (16S rRNA)
50S 33 36 L1–L36a 2 (5S and 23S rRNAs)
aThe L1 to L36 protein designations do not correspond to 36 different proteins. The protein originally designated L7 is a modified form of
L12, and L8 is a complex of three other proteins. Also, L26 proved to be the same protein as S20 (and not part of the 50S subunit). This
gives 33 different proteins in the large subunit. There are four copies of the L7/L12 protein, with the three extra copies bringing the total
protein count to 36.
26
Structure of Ribosomes in
Bacteria and Yeast

27
 rRNAs Have Complex Secondary
Structures

• The three ss rRNAs of

E.coli have specific 3-D
structure with
extensive intrachain
base pairs.
• Shape of rRNAs are
highly conserved.

28
Ribosomes in Bacteria and Eukaryotes
• Overall, very similar
• Still two subunits with
mRNA running between
• In eukaryotes, larger
(80S), more complex,
contain > 80 proteins
• Chloroplasts and
mitochondria have
ribosomes simpler than
those in bacteria.

29
 Characteristics of tRNAs

• ssRNA of 73–93 nucleotides in both bacteria

and eukaryotes
• Cloverleaf structure in 2-D
• “Twisted L” shape in 3-D
• Most have G at 5’-end; all have CAA at 3’-end
• Have modified bases
– methylated bases, and so on

30
 Characteristics of tRNAs
• Amino acid arm
– has amino acid esterified via
carboxyl group to the 2’-OH or 3’-
OH of the A of the terminal CAA
codon
• Anticodon arm
• D arm
– contains dihydrouridine (D)
– contributes to folding
• TC arm
– contains pseudouridine ()―has
bonding between base and ribose
– helps in folding 31
3-D Structure of Yeast tRNAPhe

32
 Recall: 5 Stages of Protein Synthesis
1. Activation of amino acids
– tRNA aminoacylated
2. Initiation of translation
– mRNA and aminoacylated tRNA bind to ribosome
3. Elongation
– cycles of aminoacyl-tRNA binding and peptide bond
formation…until a STOP codon is reached
4. Termination and ribosome recycling
– mRNA and protein dissociate, ribosome recycled
5. Folding and posttranslational processing
– catalyzed by a variety of enzymes
33
 Five Stages of Protein Synthesis
Stage 1 – Activation of Amino Acids

• Step 1 − creation of aminoacyl intermediate

– Aminoacyl-tRNA synthetases esterify 20 amino
acids to corresponding tRNAs.
• COO– of amino acid attacks phosphate of ATP 
creates aminoacyladenylate intermediate
– Pyrophosphate (PPi) is also cleaved, so the
reaction is driven forward by two
phosphoanhydride bond cleavages.
– The fate of the aminoacyladenylate varies.

34
Aminoacylation of tRNA

35
 Five Stages of Protein Synthesis
Stage 1 – Activation of Amino Acids

• Step 2 – transfer of aminoacyl to tRNA

– Aminoacyl-tRNA synthetases (two classes)
transfer aminoacyl group from enzyme to tRNA.
• 2’-OH or 3’-OH of tRNA attacks phosphate of
aminoacyl intermediate, creating phosphodiester bond
between amino acid and tRNA

36
Aminoacylation of tRNA

37
 Aminoacyl-tRNA Synthetases
• Each enzyme binds a specific
amino acid and the matching
tRNA.
• Most cells contain 20 different
aminoacyl-tRNA synthetases,
one for each amino acid.
• Some cells contain fewer than
20 synthetases; in this case,
one amino acid is converted to
another after charging the
tRNA.
38
 The Second Genetic Code
• Aminoacyl-tRNA synthetases must be specific for both
amino acid and tRNA.
– Matching each amino acid with correct tRNA can be viewed
as the “second genetic code.”
– The “code” is in molecular recognition of a specific tRNA
molecule by a specific synthetase.
• Only a few nucleotides in tRNA confer the binding
specificity.
– anticodon region
– The primary determinant in Ala-tRNA is a single G=U in the
amino acid arm.
39
Nucleotide Positions in tRNAs That Are Recognized by
Aminoacyl-tRNA Synthetases:
(Orange and Green Points)

40
The G-U Pair of tRNAAla Required for Recognition by
Ala-tRNA Synthetase and the Synthetic Helix That Is
Also Aminoacylated Specifically with Ala

41
 Five Stages of Protein Synthesis
Stage 2: Initiation (Prokaryotes)
• The first tRNA is unique.
• The first codon of any peptide is AUG (Met).
• All organisms have two tRNAs for Met.
– In bacteria, plus chloroplasts and mitochondria, initiation
tRNA inserts N-formylmethionine (uses a special tRNAfMet).
– Interior Met is inserted with normal tRNAMet.
• Eukaryotic protein begins with Met, not fMet, but a
special tRNA is still used for peptide initiation.

42
 Five Stages of Protein Synthesis
Stage 2: Initiation
Unusual Amino Acids Found in Peptides
• Twenty genetically encoded amino acids are common
in all organisms.
• But two additional amino acids are also found in a few
proteins and are genetically coded.
• Selenocysteine
– formed after charging an UGA(stop)-recognizing tRNA with
serine in both bacteria and eukaryotes
– this unique tRNA found at low levels in the cell
• Pyrrolysine
– directly attached to its tRNA that recognizes UAG(stop)
43
codon by some archae
Unusual Amino Acids Found in Peptides

44
 Five Stages of Protein Synthesis
Stage 2: Initiation (Prokaryotic)
• Initiation requires a large assembly.
• In bacteria, initiation requires:
– 30S ribosomal subunit
– mRNA
– fMet-tRNA
– initiation factors IF-1, IF-2, and IF-3
– GTP
– 50S ribosomal subunit
– GTP
– Mg2+
45
 Five Stages of Protein Synthesis
Stage 2: Initiation (Prokaryotic)
• Step 1: The 30S ribosomal subunit binds IF-1,
IF-2, and IF-3 and mRNA.
– Initiation factor IF-3 keeps 30S and 50S subunits
apart.
– The initiating (5’)-AUG codon of mRNA is guided to
its correct position by the Shine−Dalgarno
sequence (region in mRNA that is complementary
to a sequence in ribosomal RNA).

46
 Five Stages of Protein Synthesis
Stage 2: Initiation
• Step 2: fMet-tRNAfMet
joins the complex.
– Formylmethionine tRNA
binds to the peptidyl (P)
site along with
initiating(5’)AUG.

47
 Five Stages of Protein Synthesis
Stage 2: Initiation
• Step 3: 50S subunit
associates
– Large 50S subunit
combines with the 30S
subunit forming the
initiation complex.
• IF-2 hydrolyzes GTP.

48
mRNA Sequences That Serve as Signals for
Initiation of Protein Synthesis in Bacteria

49
 Five Stages of Protein Synthesis
Stage 2: Initiation (Eukaryotes)

• Use more initiation factors

– over 12, including eIFIA and eIF3 (functional
homologs of IF-1 and IF-3)
• Has different mechanistic details
• Has a step that circularizes the mRNA during
initiation

50
 Five Stages of Protein Synthesis
Stage 2: Initiation (Eukaryotes)
• Step 1: 40S subunit
joins with eIF1A and
eIF3
– eIF1 binds to the E site,
eIF1A binds to the A
site, EIF3 blocks the A
site preventing tRNA
binding
• Step 2: eIF2 and the
charged tRNAMet with
bound GTP join the 40S
– creating the 43S
51
preinitiation complex
 Five Stages of Protein Synthesis
Stage 2: Initiation (Eukaryotes)
• Step 3: mRNA binds
with eIF4F
– eIF4F includes
• eIF4E (binds the 5’cap)
• eIF4A (an ATPase and
RNA helicase)
• eIF4G (linker protein
which binds to PABP –
poly(A) binding
protein at the 3’ poly
(A) tail)
52
 Five Stages of Protein Synthesis
Stage 2: Initiation (Eukaryotes)
• Step 4: scanning of
the mRNA until an
AUG codon is
found
• Step 5: 60S subunit
associates and
many of the
initiation factors
are released.
53
 Five Stages of Protein Synthesis
Stage 3: Elongation (Prokaryotic)
Step 1: binding of the
incoming aminoacyl-tRNA
•Incoming aminoacyl-tRNA binds
first to an EF-Tu –GTP complex.
•The aminoacyl-EF-Tu-GTP
complex binds to the aminoacyl (A)
site of the 70S initiation complex.
•After GTP hydrolysis, EF-Tu-GDP
leaves the ribosome.

54
 Five Stages of Protein Synthesis
Stage 3: Elongation (Prokaryotic)
Step 2: peptide bond forms
•There are now two amino acids bound to tRNAs
positioned for joining.
– One is on the A site, the other on the P site.
•N-formylmethionyl group is transferred from its tRNA
in the P site to the amino acid in the A site.
– The reaction is catalyzed by the 23S rRNA (ribozyme).
•“Uncharged” (deacetylated) tRNAfMet is now in the P
site.
55
Formation of the First Peptide Bond

56
 Five Stages of Protein Synthesis
Stage 3: Elongation (Prokaryotic)
Step 3: translocation of
the ribosome
•The ribosome moves one
codon toward the 3’-end of
the mRNA.
– uses energy from GTP
hydrolysis
• GTP is part of EF-G
(translocase)
– leaves A site open for new
aminoacyl-tRNA
57
 Five Stages of Protein Synthesis
Stage 3: Elongation (Eukaryotic)
• Steps are similar to bacteria
• Elongation factors – eEF1α (EF-Tu), eEF1βγ (EF-Ts),
eEF2 (EF-G)
• Difference: Eukaryotic ribosomes do NOT have an E
site; the uncharged tRNAs are released from the P
site.

58
Five Stages of Protein Synthesis
Stage 4: Termination
• Signaled by a stop codon
• UAA, UAG, or UGA in the A site will
trigger the action of termination
factors (release factors) RF-1, RF-2,
RF-3.
• These help to:
– hydrolyze terminal peptide-tRNA bond
– release peptide and tRNA from
ribosome
– cause subunits of ribosome to dissociate
so that initiation can begin again
59
 Ribosome Rescue
• Damaged mRNA lead to the
formation of an incomplete or
peptide and a “nonstop
complex.”
• The ribosome is rescued by
trans-translation.
– A transfer-messenger RNA (tmRNA)
and small protein B (SmpB) bind to
the stalled complex in the empty A
site.
– Translation continues until a stop
codon is found in the tmRNA.
– The defective mRNA and
polypeptide are both degraded. 60
 Features of Protein Synthesis
• Large energy cost
• Can be rapid when
accomplished on clusters
of ribosomes called a
polysome
• In bacteria, tightly
coupled to transcription
– Translation can begin
before transcription is
finished.

61
 Five Stages of Protein Synthesis
Stage 5: Posttranslational Modifications
• Some proteins require modification before the fully
active conformation is achieved.
• Posttranslational modifications include:
– enzymatic removal of formyl group from first residue, or
removal of Met and sometimes additional residues
– acetylation of N-terminal residue
• Removal of signal sequences or other regions
• Attaching carbohydrates
• Removing sequence to activate an enzyme

62
 Posttranslational Modifications
• Modifying amino acids with
additional phosphates, carboxylic
acid groups, and so on
• Addition of isoprenyl groups (such
as farnesyl pyrophosphate) from
intermediates of cholesterol
synthesis pathway
– Isoprene or derived group helps
anchor proteins in membranes.
• Adding prosthetic groups
• Forming disulfide links

63
More Modified Amino Acids

64
 Ribosome Profiling
• It is a method in which a
researcher is able to isolate
mRNA currently being
transcribed.
• mRNA is isolated and
subjected to RNase; only
transcripts bound to
ribosomes will survive.
• RT-PCR is done and the
resultant cDNA is sequenced.
• The data are both
quantitative and qualitative.
65
Antibiotics and Toxins Targeting Translation
• Almost every step in
translation can be inhibited
with an antibiotic.
• Example: puromycin
– made by the mold Streptomyces
alboniger
– similar structure to 3’-end of
aminoacyl-tRNA
– so it binds to the A site of
ribosomes, forming bond with
growing peptide
– but can’t participate in
translocation and dissociation
• Thus, puromycin terminates
protein synthesis.
66
Antibiotics and Toxins Targeting Translation
• Tetracyclines
– They block the A site on the
ribosome.
• Chloramphenicol and
cycloheximide
– They block peptidyl transfer.
– Chloramphenicol inhibits
mitochondrial and chloroplast
ribosomes as well as bacterial.
• Streptomycin
– It causes code to be misread
and inhibits initiation at high
concentrations. 67
Antibiotics and Toxins Targeting Translation

• Diptheria toxin
– catalyzes ADP-ribosylation of dipthamide (modified
histidine) residue of eEF2 and inactivating it

• Ricin
– toxic protein from castor bean
– inactivates the 60S subunit by depurinating a specific
adenosine residue of the 28S rRNA

68
 Protein Targeting and Degradation
• Proteins move from site of synthesis to:
– exit a cell
– become part of the membrane
– enter a subcellular compartment, and so on
• Most have a signal sequence at or near N-terminus
– 13−36 amino acid residues in length
• This takes place in eukaryotic cells, where
subcellular organization aids in compartmentalizing
metabolic pathways.

69
 Peptides Directed to the ER
• As peptide emerges from the ribosome, the signal
sequence is bound by signal recognition particle (SRP).
• SRP/ribosome/RNA complex is delivered to the ER
lumen.
– Some modification takes place here (glycosylation, etc.).
• Transport vesicles then take proteins to Golgi
apparatus.
– Proteins are sorted here in ways poorly understood.
• Proteins enter the biosynthetic/secretory pathway.
• Proteins for mitochondria and chloroplast bind
chaperone proteins in the cytosol and are delivered to
receptors on the exterior of the organelle. 70
Directing Peptides to the ER and Beyond

71
 Glycosylation of Proteins

• Glycoproteins form by linking an

oligosaccharide to a side group on the peptide.
• N-linked (nitrogen of Asn or Arg)
– begins in the RER
• O-linked (oxygen of Ser, Thr, Tyr)
– typically occurs in the golgi or cytosol for proteins
not entering the ER
• Proteins are sorted in the Golgi and sent on to
their final destinations.
72
Glycosylation of Proteins

73
 How Proteins Are Targeted for and
Imported into the Nucleus
• Proteins for the nucleus have a nuclear localization
sequence (NLS).
– An NLS is not cleaved after the protein is targeted.
• The nuclear envelope can degrade and proteins will need to re-
enter the nucleus.
• It binds importin  and  and a GTPase called Ran.
• The complex docks at a pore and is imported.
• Ribosomal proteins are synthesized in the cytosol,
imported back into the nuclease, assembled into
subunits in the nucleolus, and then exported back to
the cytosol.
74
Targeting of Nuclear Proteins

75
 Protein Degradation Is Inevitable

• Half-lives of proteins range from seconds to

days to even months.
– Hemoglobin is long lived.
– Defective proteins are short lived, as are many
metabolism regulatory proteins that respond to
rapidly changing needs.
• But all are eventually degraded.

76
 Mechanisms of Degradation
• In E. coli, Lon (for “long form,” an ATP-dependent
protease) hydrolyzes defective or short-term
peptides.
• In eukaryotes, proteins are linked to the protein
ubiquitin.
– via activating enzyme E1, conjugating enzyme E2, and
ligating enzyme E3
• Ubiquinated proteins are cleaved by the 26
proteasome complex.
• Ubiquitin is very highly conserved among all
eukaryotes.
77
Attachment of Ubiquitin to Target Proteins

78
 Chapter 27: Summary
In this chapter, we learned that:
• amino acids are coded for on mRNA, carried by charged tRNA, and
linked together by rRNA/ribosomes
• the genetic code is universal
• there are five stages of protein biosynthesis
• ribosomes are composed of rRNA and proteins, and their sizes vary
between eukaryotes and prokaryotes
• helper proteins aid in initiation, elongation, and termination of
peptide synthesis
• polysomes are found only in prokaryotic organisms
• protein synthesis can be inhibited by antibiotics or toxins
• proteins are targeted to specific places through an N-terminal
sequence
• proteins may be posttranslationally modified
79
• all proteins are eventually recycled
80