P1251328605ijrAZ - PowerPoint PPT Presentation

Title: P1251328605ijrAZ


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Proteins Synthesis - Translation How amino
acids are linked up to form a protein. Synthesis
of proteins is a process of informa- tion
conversion from mRNA sequence to amino acid
sequence.  
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Codon dictionary Due to different combination
of four RNA bases, there are 64 different
codons. Among the 64 codons, there are three
stop codons and one start codon. Sequence a
between start codon in the beginning and a stop
codon at the end open reading frame.

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• Protein synthesis occurs on a ribosome, which
  provides a platform for protein synthesis.
• You can think of a ribosome as a tape player,
• where the tape is mRNA.
• Ribosome moves along mRNA, recruits proper
• amino acids, and catalyzes the formation of
• peptide bonds between a.a. to form a peptide
• chain.

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The mechanism that links mRNA and amino acids
together is tRNA which works as an adapter to
recognized the appropriate codon on mRNA.

• Anticodon on tRNA is complementary to
• codon on mRNA.
• 3 end of tRNA is the binding site for the
• corresponding amino acids.

•  

5
Arg
3 end
5
5 3
CGC
AUG
UAA
(Arg)
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The first step of protein synthesis is the
attachment of an amino acid to the specific tRNA
activation, which is catalyzed by aminoacyl
tRNA synthetases, a family of 20 members.

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• Then the aminoacyl tRNA approaches to
• mRNA.
• Anticondon will base pair with the
• corresponding codon on mRNA.
•  
• Ribosome works as a coordinator for the
• whole event.

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Where protein synthesis begins

• It has been verified that protein synthesis
  begins at the amino terminus.

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Steps in protein synthesis

• Step One Initiation
• mRNA associates with ribosome
• The initiation codon on mRNA, AUG, is
  thenrecognized by the first aminoacyl tRNA,
• methionyl-tRNAinit or met-tRNAinit, which
• binds to the P (peptidyl) site on ribosome.

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Methionyl tRNA or Met-tRNAinit
Met
P site
A site
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• Step Two - Chain elongation
• The second aminoacyl-tRNA enters A
• (aminoacyl) site of ribosome.
• Peptide bond is formed between the first
• a.a. (Met at P site) and the second a.a.
  at
• A site.
• Peptide bond formation is catalyzed by
• peptidyl transferase.

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Peptide bond
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• In the mean time the bond between the 1st a.a.,
  Met and tRNAinit is cleaved. First tRNA is
  released from P site.
• The dipeptide is shoveled from A site to P site
  so that A site is available for the new
  aminoacyl-tRNA to enter.
• In the mean time the ribosome is moving down on
  mRNA or the mRNA advances through the ribosome -
  translocation.

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• Step Three Termination
• Three termination codons, UAG, UAA, UGA
• do not specify any amino acids and have no
• complementary tRNAs.
• When these three codons are encountered,
  translation is terminated.
• The ribosome complex then falls apart into
• the original subunits.

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Protein synthesis and energy

• The energy requirements for synthesis are quite
  high.
• Two anhydride bonds in ATP are cleaved on
  activation of each amino acid and synthesis of an
  aminoacyl-tRNA.
• One GTP is required for entry of each amino acid
  into the ribosomal unit.
• One GTP is required during each translocation
  step.

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Fig. 4.11 Aspartame or Nutrasweet.
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Post-translational processing of
proteins(p320-p329)

• Protein synthesis establishes the primary
  structure for a protein.
• Additional processing is required to convert it
  to its biologically active form.
• This may include
• Folding
• Chemical modification

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Protein folding

• ? helix and ?-sheets fold into a tertiary
• structure.
• The purpose is to form a stable 3-D structure
• and to form maximum chemical bonding
• between side chains.  
• Protein folding is catalyzed by chaperone
• proteins.

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• Possible side chain interactions
• Hydrophobic interaction
• Ionic attractions
• Covalent bonding

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•  
• Chemical modifications
• Proteolytic cleavage
• Phosphorylation
• The attachment sites of P groups are often at
  serine, threonine and tyrosine residues.
• Attachment of carbohydrates (glycosilation)
• Attachment of prosthetic groups
• Heme, FAD, biotin, metal complex ...

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Protein Targeting

• Most proteins are synthesized by the ribosomes in
  the cytoplasm.
• However, they may be required in other cellular
  regions and organelles
• Protein targeting deals with the process of
  sorting out and moving proteins to where they are
  needed.

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Nascent protein
Endoplasmic reticulum
Nucleus
Peroxisome
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• I. Secretory and membrane proteins must be first
  transported into endoplasmic reticulum (ER)
• These proteins are produced with an extra
  sequence of 15-36 amino acids at the amino
  terminus signal peptide (sequence).
• The sequence marks it for transport to ER.
• The sequence leads the protein into endoplastic
  reticulum and is removed by hydrolysis upon
  arrival.

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• SRP (signal recognition particle) binds to the
  signal
• sequence
• 2. SRP, peptide chain and ribosome bind to SRP
• receptor on ER membrane
• 3. SRP dessociate from peptide chain signal
  sequence
• binds to the translocon (a channel) and makes it
  open.
• 4. Signal sequence and the adjacent peptide
  chain enter
• the translocon
• Signal sequence is cleaved and the peptide chain
• continue to elongate and extrude into the ER.
• (protein folding )

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SRP
SRP receptor
Gate
Translocon Translocon closed opened
Hsc 70
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• Protein transport to nucleus
• Proteins including histones, DNA and RNA
  polymerases,
• transcriptional factors etc. are synthesized in
  sytoplasm and
• imported into the nucleus.
• All these proteins contain a nuclear-localization
  
• signal (NLS), which is a seven amino-acid
• oligopeptide Pro-Lys-Lys-Arg-Lys-Val (mostly
• basic), first found in SV40 (simian virus 40)
• large T antigen.
• Large T antigen was found in the nucleus of the
  host
• cell after virus infection.

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Research case Fusion protein of pyruvate
kinase (a sytoplasmic enzyme) and NLS was found
to be expressed in the nucleus.
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Left pyruvate kinase is normally expressed in
cytoplasm. Right Chimeric pyruvate kinase
containing SV40 NLS in the N terminus is found
in the nucleus.
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• Materials needed for nuclear transport
• NLS
• - Importin ?
• - Importin ?
• - Ran (a GTPase)
• - ATP

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Ran
Ran
Transport mechanism not very well known.
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III. Protein transport to mitochondria Mitochondr
ia contain many proteins including citric acid
cycle enzymes, DNA and RNA polymerases,
cytochromes, ATP/ADP translocase , which are
synthesized in cytosol and transported to the
mitochondria. All mitochondria destined proteins
contain uptake-targeting sequences, which can be
recognized by specific receptors on mitochondria
membrane.
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Newly synthesized precursor proteins first bind
to mitochondrial-import stimulation factors
(MSF) to remain unfolded (cost ATP). The
protein-MSF complex then binds to the cell
surface receptors. First Tom37/Tom70, then
Tom20/ Tom22, and finally Tom40, a protein
channel.
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Nascent precursor protein
The uptake of mitocondrial proteins requires
energy.
Tom 40
Tom 40
Tom70 Tom 37
Tom20 Tom 22
Tom 44
Tom 37/ Tim 17
Hsc 70
Hsc 60
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• Steps in mitochondrial protein transport
• Precursor protein (a peptide chain) binds to MSF
• MSF hydrolyzes ATP to maintain peptide unfolded
• MSF-peptide binds to receptors Tom37/Tom70
• MSF is released, peptide binds to Tom20/Tom22
• Tom20/Tom22 linked to Tom40 (a transport channel)
• and peptide pass through the channel
• Peptide chain pass through Tim 23/17 on the inner
  
• membrane
• 7. Protein folding

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IV. Protein transport to chloroplasts Example
small unit of Rubisco is synthesized in
... Stromal-import sequence on N-turm of the
precursor protein first binds to receptors Toc34,
Toc 75 and Toc 86. Hydrolysis of GTP causes
Toc 75 to change conformation, the precursor
protein enters the stroma of chloroplast. Protein
binds to Hsc70 cheprone. Large subunit produced
and folded by Hsc60. Large and small subunits
combine.
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Rubisco S subunit precursor
Stromal-import signal
Toc 75 Toc 86
Toc 34
Hsc 70
Rubisco S subunit precursor
Rubisco
Hsc 60
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Regulation of protein synthesis

• A typical bacterial cell has about 4000 genes in
  its DNA genome.
• The human genome has an estimated 30,000 40,000
  genes (coding for 100,000 to 150,000 proteins).
• Only a small fraction of these genes is used by a
  cell at any given time, if at all.
• The amount of each protein generated must be
  carefully regulated to meet the needs of the cell.

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• The existence of a protein can be regulated at
  these levels
• - Transcription
• - Post-transcriptional processing (alternative
  splicing)
• - mRNA stability (poly A tails and instability
  elements)
• - Post-translational processing
• - Protein degradation
• The major control step is at the level of
  transcription initiation.

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• Two types of gene expression.
• Expression of constitutive genes (house keeping
  genes).
• Continuous transcription which produces a
  constant level of certain proteins.
• Expression of inducible or repressible genes.
• Genes that can be activated (induced) or
  deactivated (repressed). This allows for varied
  levels of RNA.

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Principles of regulatinggene expression

• In most cells, a structure gene contains
• - Promoter region - responsible for RNA
  polymerase binding and transcription initiation.
• - Binding site for inducers (enhancers).
• - Binding site for repressors.

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• RNA polymerase activity is mediated by regulatory
  proteins of two major types.
• Activators - Bind to promoter regions or
  enhancers to stimulate the activity of RNA
  polymerase.
• Repressors. Bind to specific sequences outside
  of promoter region to inhibit the activity of RNA
  polymerase.

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RNA polymerase
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• Regulatory proteins have common, discrete DNA
  binding domains (20-100 amino acid residues).
• They bind because the domain is an exact fit for
  the outer edge of the DNA helix.
• Held together by hydrogen bonding that is not
  disruptive to DNA.

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Classes of regulatory proteins

• Helix-turn-helix motif
• About 20 amino acid residues, in two helical
  regions and a turn.

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• Zinc finger motif
• Only found in eukaryotic regulatory proteins.

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• Leucine zipper motif
• Features an ?-helix region of approximately 30
  residues. Leucine occurs as every seventh one.
  This allows two molecules of the protein to form
  a zipper like region.

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Protein degradation

• After a protein has served its purpose or
  becomes damaged, it is marked for destruction.

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• Ubiquitin pathway
• - Important route for protein labeling and
  degradation in eukaryotic cells.
• Ubiquitin
• A small protein with 76 amino acid residues.
• It is highly conserved.
• Yeast and human ubiquitin differ at only 3 of the
  76 residues.

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Ubiquitin pathway

• Ubiquitin is covalently attached via a peptide
  bond to lysine residues.
• Several ubiquitin molecules are often attached to
  a single protein.
• ATP is required for the attachment.
• Degradation then occurs via proteolytic action.

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Intracellular unwanted proteins are degraded in
proteasomes. Ubiquitin labeled proteins enter
barrel- shaped proteasomes and are degraded by
trypson and chymotrypson
Proteasome