Transcript Principles of BIOCHEMISTRY

Horton • Moran • Scrimgeour • Perry • Rawn
Principles of Biochemistry
Fourth Edition
Chapter 22
Protein Synthesis
Copyright © 2006 Pearson Prentice Hall, Inc.
Chapter 22 - Protein Synthesis
The ribosome, a
complex of RNA and
protein, is the site
where genetic
information is
translated into
protein
22.1 The Genetic Code
• Codons - three letter genetic code
(nonoverlapping)
• tRNA - adapters between mRNA and proteins
• Reading frame - each potential starting point
for interpreting the 3 letter code
Fig 22.1 Overlapping vs nonoverlapping
reading of the three-letter code
Fig 22.2 Three reading frames of mRNA
• Translation of the correct message requires
selection of the correct reading frame
Fig 22.3 Standard genetic code
Features of the Genetic Code
1. The genetic code is unambiguous. In any
organism each codon corresponds to only one
amino acid.
2. There are multiple codons for most amino acids
(code is degenerate), and synonymous
codons specify the same amino acid
3. The first two nucleotides of a codon are often
enough to specify a given amino acid
Features of the Code (continued)
4. Codons with similar sequences specify similar
amino acids
5. Only 61 of the 64 codons specify amino acids
• Termination (stop codons): UAA, UGA, UAG
• Initiation codon - Methionine codon (AUG)
also specifies initiation site for protein synthesis
22.2 Transfer RNA
A. Three-Dimensional Structure of tRNA
– Transfer RNA molecules are the interpreters
of the genetic code
– Every cell must contain at least 20 tRNA (one
for every amino acid)
– Each tRNA must recognize at least one
codon
– tRNAs have a “cloverleaf” type secondary
structure with several loops or arms
Cloverleaf Secondary Structure of tRNA
• Figure 22.4 (next slide)
• Watson-Crick base pairing (dashed lines)
• tRNA has an acceptor stem and four arms
• Conserved bases (gray)
Fig. 22.4
• Cloverleaf
structure of
tRNA
Fig 22.5 Tertiary structure of tRNA
tRNA Arms
• Acceptor stem - amino acid becomes covalently
attached to tRNA at the 3’ end of this stem
• Anticodon arm - contains the anticodon, a
three-base sequence that binds to a
complementary codon in mRNA
(continued next slide)
tRNA Arms (continued)
• TyC arm - contains thymidylate (T) and
pseudouridylate (y) followed by C
• D arm - contains dihydrouridylate (D)
• Variable arm - ranges from 3-21 nucleotides
Fig 22.6
• Structure of
tRNAPhe from
yeast
Animations
• Structure of tRNA
B. tRNA Anticodons Base-Pair
with mRNA Codons
• tRNA molecules are named for the amino acid
that they carry (e.g. tRNAPhe)
• Base pairing between codon and anticodon is
governed by rules of Watson-Crick (A-U, G-C)
• However, the 5’ anticodon position has some
flexibility in base pairing (the “wobble” position)
Table 22.1
Fig 22.7 Inosinate Base Pairs
•
•
•
•
Inosinate (I) base pairs
Inosinate often found at 5’ wobble position
I can form H bonds with A, C, or U
Anticodon with I can recognize more than one
synonymous codon
Codon-Anticodon Recognition
• Wobble allows some tRNA molecules to recognize
more than one codon
• Isoacceptor tRNA molecules - different tRNA
molecules that bind the same amino acids
• Isoacceptor tRNAs identified by Roman numerals
or codons: tRNAIAla, tRNAIIAla or tRNAGCGAla
• Bacteria have 30-60 different tRNAs, eukaryotes
have up to 80 different tRNAs
Fig 22.8 Base pairing at the wobble position
22.3 Aminoacyl-tRNA Synthetases
• Aminoacyl-tRNA - amino acids are covalently
attached to the 3’ end of each tRNA molecule
(named as: alanyl-tRNAAla)
• Aminoacyl-tRNA synthetases catalyze reactions
• Most species have at least 20 different
aminoacyl-tRNA synthetases (1 per amino acid)
• Each synthetase specific for a particular amino
acid, but may recognize isoacceptor tRNAs
A. The Aminoacyl-tRNA Synthetase Reaction
• Aminoacyl-tRNAs are high-energy molecules
(the amino acid has been “activated”)
• The activation of an amino acid by aminoacyltRNA synthetase requires ATP
Amino acid + tRNA + ATP
Aminoacyl-tRNA + AMP + PPi
Fig 22.9
Fig 22.9 (cont)
B. Specificity of Aminoacyl-tRNA Synthetase
• Attachment of the correct amino acid to the
corresponding tRNA is a critical step
• Synthetase binds ATP and the correct amino
acid (based on size, charge, hydrophobicity)
• Synthetase then selectively binds specific
tRNA molecule based on structural features
• Synthetase may recognize the anticodon as
well as the acceptor stem
Fig 22.10 Structure of E. coli tRNAGln
bound to the synthetase
C. Proofreading Activity of AminoacyltRNA Synthetases
• Some aa-tRNA synthetases can proofread
• Isoleucyl-tRNA synthetase may bind valine
instead of isoleucine and incorporate it into
valyl-adenylate
• The valyl-adenylate is usually then hydrolyzed
to valine and AMP so that valyl-tRNAIle does
not form
Fig 22.11 Model of substrate-binding
site in isoleucyl-tRNA synthetase
• Ile-tRNA binds to Ile about 100x better than Val
even though they have similar size and charge
22.4 Ribosomes
• Protein synthesis is carried out by a complex
composed of the ribosome, accessory protein
factors, mRNA and charged tRNA molecules
• Initiation complex assembles at first mRNA
codon, and disassembles at termination step
• Ribosome complex moves 5’
mRNA
• Polypeptide is synthesized in N
3’ along template
C direction
A. Ribosomes Are Composed of
Both rRNA and Protein
• All ribosomes contain two subunits of
unequal size
• E. coli: 70S composed of a 30S and a 50S
• Eukaryotes: 80S composed of a 40S and a 60S
Fig 22.12 Comparison of prokaryotic and
eukaryotic ribosomes
Fig 22.13
• Assembly of the 30S ribosomal
subunit and maturation of the
16S rRNA (E. coli)
• Ribosomal proteins (6-7) bind to
16S rRNA as it is being
transcribed forming a 21S
particle
• Processing and binding of other
ribosomal proteins completes
the mature 30S subunit
Fig 22.14 Structure of the 30S ribosomal
subunit (T. thermophilus)
B. Ribosomes Contain Two
Aminoacyl-tRNA Binding Sites
• Ribosome must align two charged tRNA
molecules so that anticodons interact with
correct codons of mRNA
• Aminoacylated ends of the tRNAs are positioned
at the site of peptide bond formation
• Ribosome must hold both mRNA and growing
polypeptide chain
Fig 22.15 Sites for tRNA binding in ribosomes
22.5 Initiation of Translation
• The translation complex is assembled at the
beginning of the mRNA coding sequence
• Complex consists of:
Ribosomal subunits
mRNA template to be translated
Initiator tRNA molecule
Protein initiation factors
Fig 22.3 Standard genetic code
A. Initiator tRNA
• First codon translated is usually AUG
• Each cell contains at least two methionyl-tRNAMet
molecules which recognize AUG
• The initiator tRNA recognizes initiation codons
• Second tRNAMet recognizes only internal AUG
• Bacteria: N-formylmethionyl-tRNAfMet
• Eukaryotes: methionyl-tRNAiMet
Fig 22.16 Structure of fMet-tRNAfMet
B. Initiation Complexes Assemble
Only at Initiation Codons
• Ribosome must recognize protein synthesis start
• In prokaryotes, the 30S ribosome binds to a
region of the mRNA (Shine-Dalgarno sequence)
upstream of the initiation sequence
• S-D sequence also binds to a complementary
base sequence at the 3’ end of the 16S rRNA
• Double-stranded RNA structure binds mRNA to
the ribosome
Fig 22.17 (a) Shine-Dalgarno
sequences in E. coli mRNA
• Ribosome-binding sites at the 5’ end of mRNA for
several E. coli proteins
• S-D sequences (red) occur immediately upstream
of initiation codons (blue)
Fig 22.17 (b)
• Complementary base pairing of S-D sequence
C. Initiation Factors Help Form
Initiation Complex
• Initiation factors are required to form a
complex
• Prokaryote factors: IF-1, IF-2, IF-3
• Eukaryote factors: eIFs (8 or more factors)
Fig. 22.18 Formation of the
prokaryotic 70S initiation factor
Fig. 22.18 Formation of the
prokaryotic 70S initiation factor
Fig 22.18 (cont)
D. Translation Initiation in Eukaryotes
• Eukaryotic initiation factor 4 (eIF-4), (or cap
binding protein, CBP) binds to the (5’ end) 7methylguanylate cap of eukaryotic mRNA
• A preinitiation complex forms (40S ribosome,
aminoacylated initiator tRNA, other factors) and
searches the mRNA 5’ 3’ for an initiator codon
• The Met-tRNAiMet binds to AUG, and the 60S
ribosomal subunit binds to complete the complex
22.6 Chain Elongation is a
Three-Step Microcycle
• The initiator tRNA is in the P site
• Site A is ready to receive an aminoacyl-tRNA
• Elongation is a three-step cycle:
(1) Positioning the correct aa-tRNA in site A
(2) Formation of a peptide bond
(3) Shifting mRNA by one codon
Fig 22.19 Coupled transcription and
translation in bacteria
• Gene is being transcribed left to right
• Ribosomes bind to 5’ end of mRNA
A. Elongation Factors Dock an
Aminoacyl-tRNA in the A Site
• Bacterial elongation factor EF-Tu helps the
correct aa-tRNA insert into site A
• An EF-Tu-GTP complex binds to all aa-tRNA
molecules except fMet-tRNAfMet (initiator)
• A ternary complex of EF-Tu-GTP-aa-tRNA binds
in the ribosomal A site
• If the anticodon of the aa-tRNA correctly base
pairs with the mRNA codon, complex is stabilized
Fig 22.20 EF-Tu binds tRNAs
• EF-Tu binds to acceptor
end of aminoacylated
tRNA (Phe-tRNAPhe)
• Phe residue (green)
Fig 22.21 Insertion of
aa-tRNA by EF-Tu
during chain elongation
Fig 22.22 Cycling of EF-Tu-GTP
Fig 22.22 Cycling of EF-Tu-GTP
Fig 22.22 (cont)
B. Peptidyl Transferase Catalyzes
Peptide Bond Formation
• Peptidyl transferase activity is contained
within the large ribosomal subunit
• Substrate binding site in 23S rRNA and
50S ribosomal proteins
• Catalytic activity from 23S rRNA
(an RNA-catalyzed reaction)
Fig. 22.23 Formation of a peptide bond
C. Translocation Moves the Ribosome
by One Codon
• Translocation step: the new peptidyl-tRNA is
moved from the A site to the P site, while the
mRNA shifts by one codon
• The deaminoacylated tRNA has shifted from
the P site to the E site (exit site)
• Binding of EF-G-GTP to the ribosome
completes translocation of peptidyl-tRNA
Fig 22.24
• Translocation
during protein
synthesis in
prokaryotes
Fig 22.24
• Translocation
during protein
synthesis in
prokaryotes
Fig 22.24
(cont)
Formation of the Peptide Chain
• Growing peptide chain extends from the
peptidyl-tRNA (P site) through a tunnel in the
50S subunit
• Newly synthesized polypeptide does not begin
to fold until it emerges from the tunnel
• Elongation in eukaryotes is similar to E. coli:
EF-1a - docks the aa-tRNA into A site
EF-1b - recycles EF-1a
EF-2 - carries out translocation
Animations
• tRNA binding ribosomes
22.7 Termination of Translation
• E. coli release factors: RF-1, RF-2, RF-3
• Translocation positions one of three
termination codons in A site: UGA, UAG, UAA
• No tRNA molecules recognize these codons
and protein synthesis stalls
• One of the release factors binds and causes
hydrolysis of the peptidyl-tRNA to release the
polypeptide chain
Animations
• Protein synthesis
22.8 Protein Synthesis is
Energetically Expensive
• Four phosphoanhydride bonds are cleaved for
each amino acid added to a polypeptide chain
Amino acid activation: Two ~P bonds
ATP
AMP + 2 Pi
Chain elongation: Two ~P bonds
2 GTP
2 GDP + 2 Pi
Box 22.1 Some Antibiotics Inhibit
Protein Synthesis
• Some antibiotics prevent bacterial growth by
inhibiting the formation of peptide bonds
• Puromycin (next slide) resembles the 3’ end of
an aminoacyl-tRNA, and can enter the A site of
a ribosome
• The peptidyl-puromycin formed is bound weakly
in the A site and dissociates terminating protein
synthesis
22.9 Regulation of Protein Synthesis
A. Ribosomal Protein Synthesis Is
Coupled to Ribosome Assembly in E.
coli
– Synthesis of ribosomal proteins is tightly
regulated at the level of translation
– Ribosomal protein genes encode one
ribosomal protein that inhibits translation of
its own polycistrionic mRNA by binding near
the initiation codon of the mRNA
Fig 22.25 Comparison of proposed
secondary structures of S7-binding sites
(a) S7 site on 16S rRNA
(b) S7 site on the str mRNA
S7 protein inhibits translation by binding to the
str mRNA molecule
B. Globin Synthesis Depends on
Heme Availability
• Hemoglobin synthesis requires globin chains
and heme in stoichiometric amounts
• Globin synthesis is controlled by regulation of
translation initiation
• Heme-controlled inhibitor (HCI) phosphorylates
factor eIF-2 which then cannot participate in
translation initiation
• High heme levels interfere with HCI so that
globin synthesis proceeds
Fig 22.26
• Inhibition of
protein
synthesis by
phosphorylation of eIF-2
HCI: heme-controlled inhibitor
GEF: guaninenucleotide exchange factor
C. The E. coli trp Operon Is Regulated
by Repression and Attenuation
• The trp operon in E. coli encodes the proteins
necessary for tryptophan biosynthesis
• Because tryptophan is a negative regulator of
its own biosynthesis, synthesis can be
repressed when exogenous Trp is available
• Tryptophan is a corepressor of the trpO
operator (Figure 22.27, next slide)
Fig 22.27 Repression of the E. coli trp operon
(continued next slide)
Fig 22.27 Repression of the E. coli trp operon
(continued next slide)
Fig 22.27 (continued)
Attenuation in E. coli
• A second mechanism for regulation of the E.
coli trp operon depends on translation
• Determines whether transcription of the
operon proceeds or terminates prematurely
• GC-rich regions in the mRNA trp leader
region can base pair to form two alternative
hairpin structures which affect transcription
Fig 22.28 (a) Attenuation mechanism
for regulation
• mRNA transcript of the trp leader region contains
four GC-rich sequences which can base-pair to form
one of two alternative structures
Fig 22.28 (b)
• Structure (b) is a
pause transcription
site
Fig 22.28 (c)
• Structure (c) is
a more stable
hairpin than (b)
Transcriptional Attenuation in
the trp Operon
Transcriptional Attenuation in
the trp Operon
22.10 Posttranslational Processing
• Posttranslational modifications can occur either
before the polypeptide chain is complete
(cotranslational) or after (posttranslational)
─ deformylation of N-terminal residue (prok)
─ removal of N-terminal methionine residue
─ formation of disulfide bonds
─ cleavage by proteinases
─ phosphorylation or acetylation
Fig 22.29
• Secretory pathway in eukaryotic
cells
• Proteins synthesized in the
cytosol are transported into the
lumen of the endoplasmic
reticulum
• After further modification in the
Golgi, the proteins are secreted
Fig 22.29
• Secretory pathway in eukaryotic
cells
• Proteins synthesized in the
cytosol are transported into the
lumen of the endoplasmic
reticulum
• After further modification in the
Golgi, the proteins are secreted
(continued next slide)
Fig 22.29
(cont)
Fig 22.30 Secretory Vesicles in a maize
rootcap cell
A. The Signal Hypothesis
• Secreted proteins are synthesized by
ribosomes on the surface of the endoplasmic
reticulum
• A signal peptide is present on the N-terminus
that signals the protein to cross a membrane
• Signal peptides are 16-30 residues long, and
include 4-15 hydrophobic residues
Fig 22.31 Signal peptides from
secreted proteins
• Hydrophobic residues in blue, arrows mark sites where
signal peptide is cleaved from the precursor
Fig 22.32
• Translocation of
eukaryotic proteins
into the lumen of the
endoplasmic
reticulum
Fig 22.32
SRP: signal recognition particle
Ribophorin: ribosome receptor protein
Fig 22.32
(cont)
Fig 22.32
(cont)
B. Glycosylation of proteins
• Many integral membrane and secretory proteins
contain covalently bound oligosaccharide chains
• Carbohydrate may be from 1% to 80% of the
mass of the glycoprotein
• A common glycosylation reaction is the covalent
attachment of a complex oligosaccharide to the
side chain of an asparagine residue
Fig 22.33 Structure of a complex
oligosaccharide linked to an asparagine residue
• Man = mannose, Glc = glucose,
GlcNAc = N-acetylglucosamine