Chapter 12. Protein biosynthesis

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Chapter 12. Protein biosynthesis

• Protein biosynthesis is a process to express
  genetic information in living cells, which is
  called translation.
• The genetic information flows as
• DNA RNA
  Protein

Transcription
Translation
Reverse transcription
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1. Components of Protein Biosynthesis

• Protein biosynthesis requires amino acids, mRNA,
  tRNA, ribosomes, protein factors, and synthetic
  enzymes.
• 1) Messenger RNA a template for protein
  biosynthesis, which is read in a 5?3 direction.
  Each three nucleotides form a codon representing
  for a specific amino acid. Thus, the base
  sequence of an mRNA molecule determines the amino
  acid sequence of the protein.

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• Codons in mRNA

 
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• mRNA in eukaryotes is usually monocistronic one
  mRNA encodes only a single polypeptide chain.
• mRNA in prokaryotes usually encodes more than one
  polypeptide chain. This is called polycistronic.

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• Degeneracy of codons refers to the fact that an
  amino acid has more than one codon.
• one of the consequences of degeneracy is that a
  mutation which produces a base change in DNA may
  not result in an amino acid change in the encoded
  protein.
• Synonyms refers to the codons for the same amino
  acid. e.g. GUU, GUC, GUA, GUG represent for Val.

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• B) Universility of codons this genetic code
  system is used by all living organisms except in
  some cases
• in cytosol in
  mitochondria
• AUA Ile Met
• UGA Stop Trp
• AGA Arg Stop (animal)
• CGG Arg Trp (plant)
• CUN Leu Thr (yeast)

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• C) Reading frames refer to the different
  combinations for each three nucleotides that are
  read as a codon each mRNA sequence can be read
  in three possible reading frames.
• Reading frame 1 UUA UGA GCG CUA AAU
• Leu Stop Ala Leu Asn
• Reading frame 2 U UAU GAG CGC UAA AU
• Tyr Glu Arg Stop
• Reading frame 3 UU AUG AGC GCU AAA U
• Met Ser Ala Lys

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• D) Open reading frames refer to the runs of
  codons that start with ATG and end with TGA, TAA,
  or TAG. The open reading frames can be used to
  predict the protein sequence encoded.

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• 2) Transfer RNAs the fidelity of protein
  biosynthesis requires tRNAs to serve as adapters
  that can recognize the correspondent codons and
  carry amino acids to the right positions in
  translation.
• Each tRNA only brings with it an amino acid, and
  recognizes and binds to a specific codon.

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Secondary structure of tRNA




T?C loop






extra arm

DHU loop





Anticodon loop
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Tertiary structure of tRNA
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Codon-anticodon interaction by base pairing
tRNA
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• Wobble base pairing base pairing between the 3
  position of the codon and 5 position of the
  anticodon may occur by a non-standard way. This
  allows one tRNA to recognize more than one codon.

Examples of wobble base pairing Anticodon wobble
position base C A G U I Codon wobble
position base G U C A C
U G U
A
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Wobble base pairing of inosine with three
nucleosides
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• 3) rRNAs and ribosomes As the site of protein
  biosynthesis, ribosome is made up of two
  subunits, one is large and another is small.

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Composition of ribosomes in eukaryotes and
prokaryotes
Eukaryotic ribosome (80S)
Prokaryotic ribosome (70S)


large subunit small subunit
large subunit small subunit

Subunit size 60S
40S 50S 30S



rRNAs 5S, 5.8S, 28S 18S
5S, 23S 16S


Proteins 49
33 35
21
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• A) Polysomes several ribosomes bind to and
  translate a single mRNA molecule simultaneously
• B) Free ribosomes ribosomes occur free in the
  cytosol, usually synthesizing proteins of
  cytosol, nucleus, mitochondria or other
  organelles
• C) Membrane bound ribosomes ribosomes bind to
  the membrane of rough endoplasmic reticulum,
  usually synthesizing secretory proteins or
  membrane proteins.

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• Polysomes

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• 4) Aminoacyl-tRNA synthetase is also called
  amino acid activating enzyme, which catalyzes the
  following reactions.

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2. Steps of Protein Biosynthesis

• The steps of protein biosynthesis include
  initiation, elongation, and termination or
  release.
• 1) Initiation Translation begins with the
  assembly of an initiation complex consisting of
  an mRNA, a ribosome, and the initiator tRNA
  (fMet-tRNAiMet or Met-tRNAiMet ) . The process
  requires a number of protein factors, known as
  initiation factors.

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Formation of the initiation complex in
eukaryotic translation
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• In prokaryotes, initiation factors IF1 and IF3
  bind to the 30S subunit while IF2 binds to
  GTPfMet-tRNAiMet. The two complexes and mRNA
  combine to form a pre-initiation complex,
  releasing IF3. The 50S subunit binds with this
  complex, with hydrolyzation of the bound GTP to
  GDP and Pi, and release of IF1 and IF2, to form a
  completed initiation complex.

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Formation of the initiation complex in
prokaryotic translation
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Prokaryotic and eukaryotic initiation factors

Prokaryotic eukaryotic
Function

IF
eIF
IF
binds to small subunit before mRNA bi
nding. eIF
assists

1
1
1
1
mRNA binding.

IF
eIF
eIF
eIF
Bind initiator tRNA, stabilize ternary
complex, cause GTP/GDP

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2a
2b
2c
exchange.

IF
eIF3 Bind to the
small subunit, assist mRNA binding, cause

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dissociation of subunits after tr
anslation.

eIF
eIF
eIF
Recognize and bind the mRNA cap, assist mRNA
binding,

4a
4b
4c
eIF
eIF
eIF
hydrolyze ATP to drive scanning for the
initiator codon.

4d
4e
4f
eIF
Promotes GTP hydrolysis and
release of other initiator factors.

5
eIF

Assists subunit dissociation.

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• 2) Elongation Elongation of polypeptide chain
  consists of a series of cycles, called ribosomal
  cycles, each of which forms a new peptide bond.
• Three steps entry, peptide bond formation, and
  translocation.

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A) Entry of aminoacyl-tRNA to the A site of
ribosome (A. in prokaryotes, B. in eukaryotes. AA
aminoacyl)
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• B) Peptide bond formation

dipeptidyl-tRNA
Peptidyl- transferase
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• C) Translocation Translocation is a process
  involves the shift of the newly formed
  peptidyl(n1)-tRNA from the A site to the P site,
  with release of the deacylated tRNA from the
  ribosome. This process is mediated by another
  elongation factor, EF-G in prokaryotes, or eEF2
  in eukaryotes.

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The translocation step in protein biosynthesis
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• D) Termination when a ribosome moves onto the
  stop codon of mRNA, the stop codon in the A site
  cannot be recognized by any aminoacyl-tRNA
  molecules. Instead, release factors interact with
  the mRNA-ribosome complex, leading to discharge
  of the newly synthesized polypeptide from the
  complex.

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Termination of protein biosynthesis
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Elongation and termination factors in prokaryotes
and eukaryotes
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(Eukaryotic)
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3. Posttranslational Processing

• Newly synthesized polypeptides usually undergo
  structural changes called posttranslational
  processing.
• The most important posttranslational processing
  modification and folding.

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• Posttranslational modification
• A) Modification of protein primary structures
• Removal of the N-terminal Met residue

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• Posttranslational processing of human
  preproinsulin

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• B) Glycosylation occurs in most membrane and
  secretary proteins, such as glycoproteins.
• Two types of glycoproteins in humans O-linked
  and N-linked. Formed in endoplasmic reticulum and
  Golgi apparatus.
• N-linked
  O-linked

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• C) Modification of protein on higher-level
  structures
• Acetylation of the amino terminus
• Acetyl-SCoA H2N-protein
  Acetyl-NH-protein HSCoA
•  Phosphorylation
• ATP
  ADP
• Protein
  kinase
• Protein
  Phosphoprotein
• 
  Phosphatase
• Pi
  H2O

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• 2) Folding of newly synthesized polypeptides
• Newly synthesized polypeptide chains usually
  undergo folding, a process that requires protein
  factors called molecular chaperones. Two types of
  molecular chaperones chaperones and chaperonins.
  
• The major function of molecular chaperones is to
  assist the correct folding of nascent polypeptide
  chains by blocking their hopeless entangling or
  insignificant intermolecular interactions.

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• Molecular chaperones belong to the heat-shock
  protein (HSP) family.
• Chaperone proteins include HSP70, HSP40, and
  GrpE.
• The binding-release cycle of chaperone proteins
  with a nascent polypeptide earns time for the
  proper folding of the unfolded polypeptide chain.
  The cycle continues until the polypeptide chain
  is folded to a native conformation.

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The binding-release cycle of a chaperon-polypeptid
e complex
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• B) Chaperonins are also heat-shock proteins.
  They participate in the folding of a variety of
  proteins by forming a cylindrical structure (a
  ring) enclosing a central cavity.
• The target polypeptide chain enters the central
  cavity of the folding machine, where it is
  properly folded and is then released. The
  entering-folding process repeats until a native
  3D structure of the protein is formed.

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A folding cycle of a polypeptide by GroEL-GroES
chaperonins in E. coli cell
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4. Protein targeting

• Protein targeting is a process in which a newly
  synthesized protein is delivered to a specific
  extracellular or intracellular location.
• Secretory proteins are first synthesized by
  ribosomes bound to the rough ER (RER), with a
  signal sequence (or called signal peptide) at the
  N-terminal end, which directs the protein to be
  delivered to its functioning place.

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Signal peptidase cleavage site
A.
Hydrophobic area
N-
Secretory protein
Signal peptide
Internal signal peptide
B.
N-
Type III integral membrane protein
Signal peptide
Stop-transfer sequence

• Signal peptides of secretory proteins. (B) Type
  III integral
• membrane proteins with signal-peptide, internal
  signal-peptide,
• and stop-transfer sequences.

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• The signal peptide directs a newly synthesized
  secretary protein to enter into the RER lumen

SRP signal recognition particle
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5. Clinical correlation of protein biosynthesis

• Protein biosynthesis is the means to express the
  genes that control metabolisms in cells. Any
  mistake occurs in protein biosynthesis may result
  in severe consequences in metabolism.

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• Molecular Diseases refer to those resulting from
  abnormal protein structures due to mutation of
  genes.
• Sickle-cell anemia a result from the replacement
  of an amino acid residue at position 6 of the
  b-chain, glutamate , by another one, valine.
• Position of b-chain 1 2 3 4
  5 6 7 8
• Hemoglobin A Val-His-Leu-Thr-Pro-Glu-Gl
  u-Lys-
• Hemoglobin S Val-His-Leu-Thr-Pro-Val-G
  lu-Lys-

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• 2) Action of some antibiotics they carry out
  antimicrobial activities via inhibition of
  protein synthesis in the microorganism, such as
  tetracyclines, streptomycin, chloramphenicol, and
  so on.

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Some antibiotic inhibitors of protein
biosynthesis
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• 3) Effect of some biological molecules
  Interferons (IFNs) are cytokines produced during
  immune response to antigens, especially to viral
  infections.
• Two functions of IFNs cause viral RNA
  degradation and inhibit protein biosynthesis in
  cells.
• IFN protein kinase
  phosphorylation of eIF-2a inhibition
  of protein biosynthesis in cells
  inhibition of the viral replication.