Chapter 12 Translation

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Chapter 12Translation
The biochemistry and molecular biology department
of CMU
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Translation
The synthesis of protein molecules using mRNA as
the template, in other word, to translate the
nucleotide sequence of mRNA into the amino acid
sequence of protein according to the genetic
codon.
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Section 1 Protein Synthetic System
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• Protein synthesis requires multiple elements to
  participate and coordinate.

• mRNA, rRNA, tRNA
• substrates 20 amino acids
• Enzymes and protein factors initiation factor
  (IF), elongation factor (EF), releasing factor
  (RF)
• ATP, GTP, Mg2

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1.1 Template and Codon

• Messenger RNA is the template for the protein
  synthesis.
• Prokaryotic mRNA is polycistron, that is, a
  single mRNA molecule may code for more than one
  peptides.
• Eukaryotic mRNA is monocistron, that is, each
  mRNA codes for only one peptide.

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polycistron
3?
5?-PPP
protein
monocistron
3?
5?-mG -
PPP
protein
Non-coding
ribosomal protein binding site
Coding region
Stop codon
Starting code
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Genetic codon

• Three adjacent nucleotides in the 5-3 direction
  on mRNA constitute a genetic codon, or triplet
  codon.
• One genetic codon codes for one amino acid.

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Genetic codon
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• Three codons for stop signal UAA, UAG, UGA.
• One codon for start signal AUG. It also codes
  for methionine.
• 61 codons for 20 amino acids.

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Properties of genetic codon

• 1. Commaless
• A complete sequence of mRNA, from the
  initiation codon to the termination codon, is
  termed as the open reading frame.

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commaless

• The genetic codons should be read continuously
  without spacing or overlapping.

spacing
overlapping
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Frameshift
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• 2. Degeneracy

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• Except Met and Trp, the rest amino acids have 2,
  3, 4, 5, and 6 triplet codons.
• These degenerated codons differ only on the third
  nucleotide.

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• 3. Wobble
• Non-Watson-Crick base pairing is permissible
  between the third nucleotide of the codon on mRNA
  and the first nucleotide of the anti-codon on
  tRNA.

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Base-pair of codon and anticodon

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• 4. Universal
• The genetic codons for amino acids are always
  the same with a few exceptions of mitochondrial
  mRNA.

• Cytoplasm
• AUA Ile
• AUG Met, initiation
• UAA, UAG, UGA termination

• Mitochondria
• AUA Met, initiation
• UGA Trp
• AGA, AGG termination

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1.2 tRNA and AA Activation
tRNA
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Activation of amino acid
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Activated amino acid
Ala-tRNAAla Ser-tRNASer Met-tRNAMet
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Summary of AA activation

• Active form
• aminoacyl-tRNA
• Activation site
• a - carboxyl group
• Linkage
• ester bond
• Activation energy
• 2 high-energy bonds

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Protein synthesis fidelity

• Aminoacyl-tRNA synthetase has the proofreading
  ability to ensure that the correct connection
  between the AA and its tRNA.
• It recognizes the incorrect AA, cleaves the ester
  bond, and links the correct one to tRNA.

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Prokaryotic Met-tRNAmet

• Prokaryotic Met-tRNAmet can be formylated to
  fMet-tRNAimet.

Met-tRNAmet N10-formyl tetrahydrofolate
formyl transferase
fMet-tRNAimet tetrahydrofolate
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Initiation tRNA

• For prokaryotes
• fMet-tRNAimet can only be recognized by
  initiation codon.
• Met-tRNAemet is used for elongation.
• For eukaryotes
• Met-tRNAimet is used for initiation.
• Met-tRNAemet is used for elongation.

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1.3 Ribosomes

• Ribosome is the place where protein synthesis
  takes place.
• A ribosome is composed of a large subunit and a
  small subunit, each of which is made of ribosomal
  RNAs and ribosomal proteins.

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Molecular components of ribosome of prokaryotes
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Ribosome of prokaryotes
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Three sites on ribosomes
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A site, P site and E site
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Section 2 Protein Synthetic Process
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General concepts

• The direction of the protein synthesized
  N-terminal?C-terminal
• The direction of template mRNA 5? 3end
• The process of Protein
• initiation
• elongation
• termination

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2.1 Initiation
Prokaryotic initiation

• Four steps
• Separation between 50S and 30S subunit
• Positioning mRNA on the 30S subunit
• Registering fMet-tRNAimet on the P site
• Associating the 50S subunit
• Three initiation factors IF-1, IF-2 and IF-3.

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Shine-Dalgarno (S-D) sequence

• -AGGA PuPuUUUPuPu AUG-
• purine rich of 4-9 nts long
• 8-13 nts prior to AUG

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Alignment of 16S rRNA
The 3end of 16s rRNA has consensus sequence UCCU
which is complementary to AGGA in S-D sequence
(also called ribosomal binding site).
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Initiation 1-2

• The IF-1 and IF-3 bind to the 30S subunit, making
  separation between 50S and 30S subunit.
• The mRNA then binds to 30S subunit.

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Initiation 3

• The complex of the GTP-bound IF-2 and the
  fMet-tRNA enters the P site.

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Initiation 4

• The 50S subunit combines with this complex.
• GTP is hydrolyzed to GDP and Pi.
• All three IFs depart from this complex.

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IF-2
Pi
-GTP
GDP
IF-3
IF-1
One GTP is consumed in initiation course?
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eukaryotic initiation

• Four steps
• Separation between 60S and 40S subunit
• binding Met-tRNAimet on the 40S subunit
• Positioning mRNA on the 40S subunit
• Associating the 60S subunit

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Eukaryotic initiation factors
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Process of eukaryotic initiation
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2.2 Elongation

• Three steps in each cycle
• Positioning an aminoacyl-tRNA in the A site---
  Entrance
• Forming a peptide bond---Peptide bond formation
• Translocating the ribosome to the next
  codon---Translocation
• Elongation factors (EF) are required.

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Step 1 Entrance

• An AA-tRNA occupies the empty A site.
• Registration of the AA-tRNA consume one GTP.
• The entrance of AA-tRNA needs to activate EF-T.

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GTP
Tu
Ts
Ts
GDP
Tu
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Step 2 Peptide bond formation

• The peptide bond formation occurs at the A site.
• The formylmethionyl group is transferred to aNH2
  of the AA-tRNA at the A site by a peptidyl
  transferase.

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Peptide bond formation 1
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Peptide bond formation 2
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Step 3 Translocation

• EF-G is a translocase.
• GTP bound EF-G provides the energy to move the
  ribosome one codon toward the 3 end on mRNA.
• After the translocation, the uncharged tRNA is
  released from the E site.

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Translocation
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fMet
fMet
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Eukaryotic elongation

• Elongation factors are EF-1 (EF-T) and EF-2
  (EF-G).
• There is no E site on the ribosome.

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2.3 Termination

• Prokaryotes have 3 release factors RF-1, RF-2
  and RF-3.
• RF-1 and RF-2 Recognizing the termination codons
• RF-3 GTP hydrolysis and coordinating RF-1/RF-2
  and rpS
• Eukaryotes have only 1 releasing factor eRF.

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Termination 1

• The peptidyl transferase is converted to an
  esterase.

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Termination 2

• The uncharged tRNA, mRNA, and RFs dissociate from
  the ribosome.

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RF
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Energy consumption
initiation one GTP (IF-2-GTP) AA activationtwo
P bonds elongation two GTP
(Tu-GTP, EF-G-GTP) termination one GT
P (RF-3) Total at least four high-energy bonds
per peptide bond are consumed.
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Translation of prokaryotes
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Polysome

• Proteins are synthesized on a single strand mRNA
  simultaneously, allowing highly efficient use of
  mRNA.

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Section 3 Protein Modification and Protein
Targeting
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3.1 Protein Folding

• The macromolecules assisting the formation of
  protein secondary structure include
• molecular chaperon
• protein disulfide isomerase (PDI)
• peptide prolyl cis-trans isomerase (PPI)

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Chaperons

• A group of conserved proteins that can recognize
  the non-native conformation of peptides and
  promote the correct folding of individual domains
  and whole peptides.
• Heat shock protein (HSP)
• HSP70, HSP40 and GreE family
• Chaperonin
• GroEL and GroES family

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Mechanism

• Protect the unfolded segments of peptides first,
  then release the segments and promote the correct
  folding.
• Provide a micro-environment to promote the
  correct native conformation of those peptides
  that cannot have proper spontaneous folding.

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Mechanism
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3.2 Modification of primary structure

• Removal of the the first N-terminal methionine
  residue
• Covalent modification of some amino acids
  (phosphorylation, methylation, acetylation, )
• Activation of peptides through hydrolysis

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3.2 Modification of spatial structure

• Assemble of subunits Hb
• Attachment of prosthetic groups glycoproteins
• Connection of hydrophobic aliphatic chains

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3.4 Protein Targeting

• The correctly folded proteins need to be
  transported to special cellular compartments to
  exert desired biological functions.
• AAs sequence on the N-terminus that directs
  proteins to be transported to proper cellular
  target sites is called signal sequence.

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Signal sequences
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a. Secretory protein
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Signal peptide

• All the secretory proteins have the signal
  peptide.
• Consist of 13-36 AA in three regions
• Positively charged AA at N-terminus
• Hydrophobic core of 10-15 AA in the medial region
• Small polar AA at C-terminus

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Signal sequence for ER
Cleavage site
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Secretory protein into ER
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b. Mitochondrial protein

• Mitochondrial proteins in cytosol are present in
  precursor forms.
• Signal sequence of 20-25 AA at N-terminus are
  rich in Ser, Thr, and basic AA.

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b. Mitochondrial protein

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c. Nuclear protein
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Section 4 Interference of Translation
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• The protein synthesis is highly regulated.
• This process can also be the primary target for
  many toxins, antibiotics and interferons.
• These interferants interact specifically with
  proteins and RNAs to interrupt the protein
  synthesis.

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Antibiotics
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Antibiotics
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Puromycin

• It has a similar structure to Tyr-tRNA.
• It works for both prokaryotes and eukaryotes.

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Toxins

• Some toxins, such as plant protein Ricin, is
  among the most toxic substance known, which acts
  on 60s subunits.

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Diphtheria toxin
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Interferon

• Interferons are cytokines produced during immune
  response to antigens, especially to viral
  infections.

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Interferon
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mRNA