Protein Synthesis, Processing, and Regulation

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Protein Synthesis, Processing, and Regulation
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Introduction

• Proteins are the active players in most cell
  processes, implementing the myriad tasks that are
  directed by the information encoded in genomic
  DNA.
• What do we know about Protein Expression?
• Proteins are synthesized from mRNA templates by a
  process that has been highly conserved throughout
  evolution.
• Translation is carried out on ribosomes, with
  tRNAs serving as adaptors between the mRNA
  template and the amino acids being incorporated
  into protein.

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Transfer RNAs

• Transfer RNAs, or tRNAs, possess unique
  identifying sequences that allow the correct
  amino acid to be attached and aligned with the
  appropriate codon in mRNA.
• Transfer RNAs are approximately 70 to 80
  nucleotides long and have characteristic
  cloverleaf structures.

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Transfer RNAs

• Aminoacyl tRNA synthetases are a group of enzymes
  that recognize a single amino acid, as well as
  the correct tRNA (or tRNAs) to which that amino
  acid should be attached.

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8.3 Nonstandard codon-anticodon base pairing

• tRNAs bind to mRNA by complementary base pairing.
• This base pairing is less stringent than normal
  A-T/G-C base pairing.

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The Ribosome

• Ribosomes are the sites of protein synthesis in
  all cells.
• Ribosomes consist of proteins subunits and rRNA
  molecules.
• Ribosomal RNAs, or rRNAs, are the ribosomal
  components of ribosomes.

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8.4 Ribosome structure

• The general structures of prokaryotic and
  eukaryotic ribosomes are similar.
• They are designated by their sedimentation rates.

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The Organization of mRNAs and the Initiation of
Translation

• Although the mechanisms of protein synthesis in
  prokaryotic and eukaryotic cells are similar,
  there are also differences.
• 5 untranslated regions are noncoding sequences
  on the 5 terminal portions of both prokaryotic
  and eukaryotic mRNAs.

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The Organization of mRNAs and the Initiation of
Translation

• The Shine-Dalgarno sequence is a specific
  sequence that aligns the mRNA on the ribosome for
  translation by base-pairing with a complementary
  sequence near the 3 terminus of 16S rRNA.

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The Process of Translation

• Translation is generally divided into three
  stages initiation, elongation, and termination.
• A number of specific nonribosomal proteins are
  also required for the various stages of the
  translation process.

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The Process of Translation

• Initiation factors are involved in the first
  translation step in both eukaryotes and
  prokaryotes.
• Initiation in eukaryotes is more complicated and
  requires at least twelve proteins, each
  consisting of multiple polypeptide chains.

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8.11 Initiation of translation in eukaryotic
cells (Part 1)
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8.11 Initiation of translation in eukaryotic
cells (Part 2)
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8.11 Initiation of translation in eukaryotic
cells (Part 3)
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The Process of Translation

• Elongation factors, which are complexed to GTPs,
  escort the aminoacyl tRNA to the ribosome.
• The next step in elongation is translocation,
  which requires another elongation factor and is
  coupled to GTP hydrolysis.

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The Process of Translation

• As elongation continues, the eEF1a, or EF-Tu,
  that is released from the ribosome bound to GDP
  must be reconverted to its GTP form.
• Release factors are proteins that recognize stop
  codons and terminate translation of mRNA.

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8.14 Termination of translation

• Release factors are proteins that recognize stop
  codons and terminate translation of mRNA.

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The Process of Translation

• Messenger RNAs can be translated simultaneously
  by several ribosomes in both prokaryotic and
  eukaryotic cells.
• A polysome, or polyribosome, is a group of
  ribosomes bound to an mRNA molecule.

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Regulation of Translation

• One mechanism of translational regulation is the
  binding of repressor proteins to specific mRNA
  sequences.
• The regulation of translation of ferritin mRNA by
  iron is similar to the regulation of transferrin
  receptor mRNA stability.

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Regulation of Translation

• Translation can also be regulated by proteins
  that bind to specific sequences in the 3
  untranslated regions of some mRNAs.
• Localization of mRNAs to specific regions of eggs
  or embryos plays an important role in development.

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8.20 Regulation of translation by
phosphorylation of eIF2 and eIF2B

• Another mechanism of regulating translation in
  eukaryotic cells is by modulating the activity of
  initiation factors such as eIF2 and eIF4e. This
  can be done by phosphorylation of the initation
  factors.

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Post-Translational Modifications

• Protein folding
• Protein Cleavage
• Glycosylation
• Attachment of Lipids
• N-myristoylation
• Prenylation
• Palmytolation
• Phosphorylation
• Ubiquitination

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Protein Folding and Processing

• The classic principle of protein folding is that
  all the information required for a protein to
  adopt the correct three-dimensional conformation
  is provided by its amino acid sequence.
• Molecular chaperones are proteins that facilitate
  the folding of other proteins.
• Two specific families of chaperone proteins act
  in a general pathway of protein folding in both
  prokaryotic and eukaryotic cells Heat shock
  proteins and Chaperonins.
• Unfolded polypeptide chains are shielded from the
  cytosol within the chamber of the chaperonin.

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8.21 Action of chaperones during translation and
Transport

• Some chaperones bind to nascent polypeptide
  chains that are still being translated on
  ribosomes, thereby preventing incorrect folding
  or aggregation of the amino-terminal portion of
  the polypeptide before synthesis of the chain is
  finished.

• Chaperones also stabilize unfolded polypeptide
  chains during their transport into subcellular
  organelles.

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Enzymes that Catalyze Protein Folding

• Protein disulfide isomerase, or PDI, catalyzes
  disulfide bond formation and plays an important
  role by promoting rapid exchanges between paired
  disulfides.
• Peptidyl prolyl isomerase is a catalyst enzyme
  that plays an important role in the folding of
  some proteins.

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Protein Cleavage

• Proteolysis is an important step in the
  maturation of many proteins and involves cleavage
  of the polypeptide chain.
• Signal sequences target many secreted proteins to
  the plasma membrane of bacteria or to the
  endoplasmic reticulum of eukaryotic cells while
  translation is still in progress.

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Protein Cleavage

• Signal peptidase is a specific membrane protease
  that cleaves the signal sequence after the
  remainder of the polypeptide chain passes through
  the channel membrane during translation.
• Active enzymes or hormones, such as insulin, form
  via cleavage of larger precursors.

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Glycosylation

• Glycosylation is a process in which many
  proteins, particularly in eukaryotic cells, are
  modified by the addition of carbohydrates.
• Glycoproteins are proteins to which carbohydrate
  chains have been added.
• Glycoproteins are classfied as either N-linked or
  O-linked, depending on the site of attachment of
  the carbohydrate side chain.

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Attachment of Lipids

• N-myristoylation is a process in which myristic
  acid is attached to an N-terminal glycine
  residue.
• Prenylation is a type of modification in which
  specific types of lipids are attached to the
  sulfur atoms in the side chains of cysteine
  residues located near the C terminus of the
  polypeptide chain.
• Palmitoylation is a type of fatty acid
  modification in which palmitic acid is added to
  sulfur atoms of the side chains of internal
  cysteine residues.

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Attachment of Lipids

• Some proteins in eukaryotic cells are modified by
  the attachment of lipids to the polypeptide
  chain.
• Glycolipids are lipids that are linked to
  oligosaccharides and then added to the C-terminal
  carboxyl groups of some proteins, where they
  serve as anchors that attach the proteins to the
  external face of the plasma membrane.
• Glycosylphosphatidylinositol, or GPI anchors, are
  glycolipids that are attached to proteins that
  contain phosphatidylinositol.

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Protein Phosphorylation

• Protein kinases catalyze protein phosphoylation
  by transferring phosphate groups from ATP to the
  hydroxyl groups of the side chains of serine,
  threonine, or tyrosine residues.

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Protein Phosphorylation

• Protein-serine/threonine kinases are protein
  kinases that phosphorylate serine and threonine
  residues.
• Protein-tyrosine kinases are protein kinases that
  phosphorylate tyrosine residues.
• Protein phosphatases act to reverse protein
  phosphorylation and catalyze the hydrolysis of
  phosphorylated amino acid residues.

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8.42 The ubiquitin-proteasome pathway

• Damaged proteins are recognized and rapidly
  degraded within cells, thereby eliminating the
  consequences of mistakes made during protein
  synthesis.
• Ubiquitin is a marker in eukaryotic cells that
  targets cytosolic and nuclear proteins for rapid
  proteolysis.
• Proteasomes are large, multi-subunit protease
  complexes that recognize and degrade
  polyubiquinated proteins.

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8.42 The ubiquitin-proteasome pathway