Translation and Proteins

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Chapter 14

• Translation and Proteins

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Translation

• Defined as the polymerization of amino acids into
  a polypeptide chain
• Occurs at/on ribosomes using mRNA templates
• tRNAs covert triplet codons into specific amino
  acid sequence by base pairing with anticodon
• Adaptor theory by Crick (1957)

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Ribosome Structure

• About 10,000 ribosomes in growing E. coli cell
  (can be more)
• Bacterial ribosomes about 250 nm across
• Made of rRNAs and ribosomal proteins
• Two subunits, large and small
• Eukaryotes and prokaryotes generally similar but
  different enough
• 70S vs. 80S, etc. (see figure 14-1)

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tRNA Structure

• 75-90 nucleotides in size
• General structure highly conserved in prokaryotes
  and eukaryotes
• Transcribed as larger precursor, processed
• Nucleotides removed
• Nucleotides added
• Bases modified (many modified bases present)

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Modified Bases in tRNAs
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tRNA Structure

• Cloverleaf model
• Convenient to show major loops and base pairing
• Actually more L shaped (figure 14-4)
• 3 loops or arms
• D arm, T?U arm, anticodon arm
• 5 end is G
• 3end is always CCA
• Amino acid attached to the terminal adenylate
• Some of these nucleotides can be added added
  posttranscriptionally

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tRNA Cloverleaf
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3D tRNA Structure

• 1974

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tRNA Charging

• Aminoacyl tRNA synthetases
• At least 20 different ones
• One recognizes each amino acid and the set of
  tRNAs required to read the codon(s) calling for
  that amino acid (isoaccepting tRNAs)
• Use ATP for energy (AMP PPi released)
• Adenylated amino acid intermediate
• Carboxyl end of amino acid connected to 3 end of
  tRNA

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tRNA Charging
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The Many Factors of Protein Synthesis

• Each phase has its own set of factors

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Phases of Translation

• Three phases
• Initiation
• Small and large subunits, charged initiator tRNA,
  GTP, Mg and several initiation factors (Ifs)
• First codon is AUG
• Shine Dalgarno box (AGGAGG in E. coli) at 10 to
  AUG
• Ends with the addition of the large subunit
• Elongation
• Termination

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Initiation
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Elongation

• Addition of large subunit creates P (or peptidyl)
  and A (aminoacyl or acceptor) sites in fully
  assembled ribosome
• Initiator tRNA binds to AUG in P site
• Incoming aminoacyl tRNA delivered to to codon in
  A site by EF-Tu, cleaving GTP
• Peptidyl transferase transfers amino acid/peptide
  on tRNA in P site to amino acid on tRNA in A site
• Enzyme function in 23S rRNA (not a protein)
• Peptide in A site translocated to P site by EFG
  using GTP energy
• E site for ejection of uncharged tRNA

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Elongation
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Termination

• UAA, UAG or UGA in A site
• GTP-dependent release factors (RFs) recognize
  these codons and cleave polypeptide from the
  terminal tRNA
• Ribosome disassembles and can be reassembled at
  new initiation site

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Termination
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Polyribosomes

• Polysomes
• Multiple ribosomes bound to a single mRNA
• Increase overall rate of protein production
• One mRNA simultaneously directs the synthesis of
  multiple copies of a polypeptide

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Polysomes

• Found in eukaryotes and prokaryotes
• These examples are from rabbit reticulocytes and
  midgefly salivary glands
• Note polypeptide chain lengthens as ribosomes
  progress along mRNA

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Eukaryotic Translation

• Larger ribosomes, larger rRNAs, for ribosomal
  proteins
• More factors
• mRNAs have longer half-life
• Hours or longer vs. minutes in prokaryotes
• Short recognition sequence surrounds initiation
  AUG (ACCAUGG, Kozak box)
• Initiation methionine not formylated

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Protein-based Inborn Errors of Metabolism

• Archibald Garrod, early 20th century (1902)
• Alkaptonuria seemed to run in families
• Alkaptons in urine turn black upon exposure to
  oxygen
• Phe or tyr in diet increase alkaptons in urine
• First to suggest that hereditary information
  controls chemical reactions in the body
• Connecting genes to enzymes (or unit factors to
  ferments as he said)
• Another man ahead of his time

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Alkaptonuria The Pathway
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Phenylketonuria

• PKU
• First described in 1934, causes MR
• Autosomal recessive disease, lack phenylalanine
  hydroxylase (about 30 in carriers)
• Can give 50X level of phe in blood
• 1/11,000 births
• Treatable by diet

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One Gene One Enzyme

• George Beadle and Edward Tatum (1933)
• Neurospora crassa auxotrophs, minimal and
  complete media
• Auxotrophs required only a single compound to
  grown

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Beadle and Tatum
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Metabolic Pathways
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Studies of Human Hemoglobin

• Sickle cell anemia
• Starch cell electrophoresis revealed a change in
  HbA and HbS
• Protein sequence analysis by Vernon Ingram
  (1954-1957) showed specific glutamic acid to
  valine substitution
• Basic for an inherited molecular disease
  established
• 1/625 African-Americans, 1/145 couples have two
  heterozygote carriers

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Hemoglobin

• Normal vs. sickled red blood cells

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Starch Gel Analyses
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Developmentally Regulated Expression of Hb Genes

• Different variations of hemoglobin molecules used
  in human (mammalian) embryo, fetal and adult
  stages

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Colinearity of Nucleotide and Amino Acid Sequences

• Yanofsky studies of E. coli trpA gene
• Mapped many mutations to gene
• Sequenced mutant proteins
• Mutation mapped positions/order of actual amino
  acid changes (couldnt sequence DNA yet)

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Nucleotide/Amino Acid Colinearity
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Protein Structure Basis for Biological Diversity

• 20 amino acids unique but can be grouped into 4
  or so groups
• Choice and order of amino acids determines
  proteins structure and thereby its function
• With 20 possibilities for each position an
  average 300 aa polypeptide has a nearly infinite
  number of possible sequences

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Amino Acids
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Protein Structure

• Primary structure
• Amino acid sequence
• Secondary structure
• Hydrogen bonding by polypeptide backbone
• Alpha helix and beta sheet
• Tertiary structure
• Interactions between R groups of amino acids of a
  polypeptide chain
• Quaternary structure
• Interactions between R groups of amino acids of
  two or more polypeptide chains

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Primary Structure

• Sequence of amino acids
• Peptide bonds
• Major impact on upper levels of protein structure

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Secondary Structure

• a-helix and b-pleated sheet proposed by Pauling
  (1951)
• Hydrogen bonding between atoms of backbone

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Tertiary Structure

• Interactions between atoms of R-groups of a
  single polypeptide chain
• But protein can still be composed of multiple
  polypeptides
• Motifs and domains

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Tertiary Structure

• Interactions between atoms of R-groups of
  different polypeptides
• subunits

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Posttranslational Modifications

• 20 different amino acids encoded by genetic code
• Hundreds of different amino acids found in
  proteins
• Examples
• N-terminal amino acid(s) commonly removed or
  modified
• Other amino acid modifications
• Phosphorylation, methylation, acetylation, etc.
• Glycosylation
• Peptide cleavage (e.g. insulin)
• Signal/targeting peptide removal
• Metal cofactor or other non-amino acid additions

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Chaperones

• Proteins required for the proper folding of other
  proteins
• Molecular chaperones or chaperonins
• Some genetic disorders caused by lack of a
  chaperonin
• Alzheimer disease and mad cow or
  Creutzfeldt-Jacob disease (prion diseases) caused
  by improper folding

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Protein Function Related to Protein Structure

• DNA and RNA can be considered vehicles to store
  and express genetic information
• Proteins are at the heart of cellular function
• Except for some RNAs with catalytic functions
• Structural, transport, defense, signals, DNA
  condensation and regulation, enzymes

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Enzymes Biological Catalysts

• Lower activation energy
• Active site
• Anabolic, catabolic or amphibolic pathways

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Protein Domains

• Made up commonly of 50-300 amino acids
• Associated with specific functions of the protein
• Catalytic domain, protein-binding domain,
  DNA-binding domain, etc.
• Tertiary structure

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Exon Shuffling

• Exons encoding domains may be mixed and matched
  through recombination and natural selection to
  produce new genes/proteins with new or modified
  functions (Walter Gilbert 1977)
• Reshuffling simplified by introns and RNA
  processing
• Human LDL receptor gene seems to be constructed
  from portions of other genes
• Ford Doolittle (1978) proposed intron early
  theory which would facilitate this type of
  evolution
• Yeast would have lost most of their introns since
  then

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Human LDL Receptor Gene

• Human LDL receptor gene has regions shared with
  other proteins