Protein Synthesis and Sorting

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Chapter 20

• Protein Synthesis and Sorting

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Translation

• 5 components of translation of mRNA to protein
• Ribosomes
• tRNA
• Aminoacyl-tRNA synthetases
• mRNA
• Protein factors

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Ribosomes

• Not functional without mRNA
• Complex of rRNA and protein
• rRNA is responsible for most of the activities of
  the ribosome
• 4 sites in the ribosome used in protein synthesis
• mRNA binding site
• A (aminoacyl) site new tRNA bringing in amino
  acid
• P (petidyl) site growing peptide on the tRNA
• E (exit) site site where tRNA leaves the
  ribosome

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Ribosome
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tRNA

• Amino acid is linked to the 3 OH on Adenine
  near the end of the tRNA
• Once the amino acid is linked to tRNA, it is an
  aminoacyl-tRNA
• AA is charged and active
• Anticodon allows the tRNA to recognize the codon
  in the mRNA
• Anticodon is written 3 to 5

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tRNA
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Wobble Hypothesis

• There is a wobble in the base pairing in the 3rd
  nucleotide so that the tRNA can be used for
  multiple codons for the same amino acid
• The 3rd base-pair is usually an inosine a
  modified nucleotide that can bind to U, A or C

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Aminoacyl tRNA Synthetases

• 20 different ones one per each amino acid
• Requires ATP to charge the tRNA with the amino
  acid
• This trapped energy will help to form the peptide
  bond later

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mRNA

• mRNA has 2 areas of nucleotide sequence that
  doesnt get converted to amino acids both
  crucial to translation
• 5 leader before the start codon AUG
• 3 trailer after the stop codon
• Different from the 5 CAP and 3 polyA tail
• Eukaryotic cells have a single mRNA for each
  polypeptide
• Prokaryotic cells have multiple genes (polygenic)
  on the mRNA that encode for proteins used in
  similar functions
• Called an operon

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Translation

• Proteins are synthesized from N terminus to C
  terminus
• mRNA is read from 5 to 3
• 3 steps in translation
• Initiation mRNA on ribosome and adding Met
• Elongation addition of amino acids based on
  codon sequence
• Termination release of mRNA and polypeptide
  from the ribosome

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Translation
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Initiation of Translation

• Initiation factor 1 (IF1), IF 2 and IF3 are
  required to bind to the small subunit of the
  ribosome
• IF is a GTP protein
• mRNA and tRNAfMet bind to small subunit as well
• Once the tRNAfMet moves to the P-site, the large
  subunit is recruited and the ribosome complex is
  formed
• The addition of the large subunit causes the
  cleavage of GTP and the release of IF2

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Inititation
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Elongation of Translation

• Binding of aminoacyl tRNA
• Requires 2 GTP that come in with the EF to help
  get the right amino acid, uses 2 GTP
• Peptide bond formation by peptidyl transferase
• Probably a function of the rRNA - ribozyme
• Translocation
• Requires GTP and elongation factor G (EF-G) to
  move to the next codon
• Peptidyl tRNA contains the growing polypeptide
  chain

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Termination of Translation

• Termination is attained when the STOP codon (UAG,
  UAA, UGA) in the mRNA
• There is no tRNA
• The STOP codon is recognized by the release
  factors
• The polypeptide is transferred to water, forming
  the free carboxyl end

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Termination
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Translational End Notes

• Protein synthesis is very energy intensive
• 2 ATP to charge the tRNA
• 3 GTP 2 for the aminoacyl tRAN and 1 for
  translocation
• Polyribosome many ribosomes along a single mRNA
  molecule
• Leads to many copies of the protein off the same
  mRNA

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Protein Folding

• The protein needs to get into the proper 3-D
  structure
• Molecular chaperones are required
• BiP (binding protein) has a hydrophobic amino
  acids that help proteins to fold by binding
  hydrophobic regions and keeping them from
  prematurely folding
• Protein disulfide isomerase forms and breaks
  disulfide bonds between cysteine residues
• May start before translation is finished and
  keeps being remodeled until most stable form

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Post-translational Modifications

• Removal of the initial Met
• Removal of signal sequence
• Cleavage of precursor protein
• Chemical modification - -CH3, -PO4, acetylation
• Glycosylation
• Bind prosthetic group
• Protein splicing removal of AA sequences called
  inteins remaining exteins spliced together to
  make mature proteins
• Single polypeptide or between 2 polypeptides

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Protein Splicing
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Protein Targeting

• mRNA moves through the nuclear pore to the free
  ribosomes
• 2 paths the ribosome may take
• If transmembrane protein, ribosome attaches to ER
  and transfer the protein to the interior of the
  ER co-translational import
• If it is to be a cytosl protein or for the
  mitochondria or chloroplast or the nuclear
  interior, the protein is made and released from
  the ribosome post-translational import

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Protein Targeting
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Co-translational Import

• The N terminus of the protein has an ER signal
  sequence which causes the ribosome to halt
  synthesis and move to the ER and sends the
  protein into the lumen of the ER and at the end
  of synthesis the signal is removed by a signal
  peptidase

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Co-Translational Import

• Ribosome makes polypeptide chain to the signal
  sequence and the SRP recognizes it and binds to
  it
• SRP is made up of 6 polypeptides and RNA and
  recognizes the signal sequence in the growing
  polypeptide
• SRP moves the ribosome to the ER and attaches to
  the tanslocon (complex of SRP receptor, ribosome
  receptor, pore protein and signal peptidase)
• GTP binds SRP-SRP receptor and translation starts
  again
• GTP is hydrolyzed and SRP is released
• Protein moves into the ER lumen
• When translation is done, signal peptidase
  cleaves off the signal and releases the ribosome

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SRP localization
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Glycoproteins

• Glycoproteins are predominant in the ER
• ER lumen
• Golgi Apparatus
• Sorts and distributes proteins to other areas
• Default Golgi to the plasma membrane secretory
  pathway
• Special signals for other areas mannose-6-PO4
  for the lysosomes
• KDEL (Lys-Asp-Glu-Leu) sequence final
  destination is the ER, Golgi binds to KDEL and
  shuttles it back to ER

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Transmembrane Proteins
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Post-Translational Import

• Import into these areas after translation
• Nuclear interior, mitochondria, chloroplast or
  peroxisome
• Signal to target it to correct organelle
• SKL (Ser-Lys-Leu) target to peroxisome
• Nuclear localization signal to the nucleus

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Mitochondria and Chloroplast

• Need to get the protein to the interior of these
  structures when made outside of the organelle
• Most proteins come form the nuclear genes
• Signal sequence is called the transit sequence
  at the N terminus
• Removed by transit peptidase usually before the
  transfer is complete
• Both hydrophobic and hydrophilic AA involved
  hydrophilic are the positively charged ones

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Transport Complexes

• Specialized transport complexes in the inner and
  outer membranes
• TOM or TOC translocase of the outer membrane
• TIM or TIC translocase of the inner membrane
• Transit sequence receptor binds the sequence and
  translocates the peptide across the membrane
• If in the inner membrane there is contact between
  the inner and outer membrane so that it can pass
  all the way through both membranes

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TOM and TIM TOC and TIC
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Movement into Mitochondria

• Protein crosses into the mitochondria through a
  small pore unfolded
• Chaperone protein, Hsp 70, binds to new protein
  in the cytosol loosely folded
• Transit sequence at N terminus binds the
  receptor component TOM at surface

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• Chaperone is released by ATP hydrolysis as the
  protein moves thru TOM and TIM

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• Mitochondrial Hsp 70 binds temporarily
• Requires ATP hydrolysis to remove chaperone,
  drives translocation also thought to be ATP in
  chloroplasts
• Additional chaperones may be required to refold
  the protein

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Target Mitochondrial Compartments

• Use N terminal transit sequence
• Hydrophobic sorting signal to target to the final
  destination
• Acts as a STOP-transfer sequence
• Embeds the protein in the membrane

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Chloroplast Targeting

• Chloroplasts may use multiple signals
• Thylakoid signal sequence to get to the thylakoid
  or the membrane
• GTP dependent protein which is similar to the
  signal recognition particle
• Removal of the thylakoid signal sequence
• Into the thylakoid space
• ATP dependant mechanism