Initiation

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Chapter 13

• Part 2

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Initiation

• Most ribosomes not participating in translation
  exist as large and small subunit
• Need all of the reagents at the left
• Small subunit binds several initiation factors
  and this complex binds mRNA, bacteria use 6
  ribonucleotides AGGAGG upstream of AUG site
  Shine-Delgarno sequence to interact with 16S rRNA
• Use another IF to enhance binding of tRNAf-met to
  AUG
• sets up reading frame, use energy in 1 GTP and IF
  are released

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Elongation

• Growing 1 peptide at time
• Once ribosome is formed places for 2 charged
  tRNA
• P or peptidyl site growing chain
• A or aminoacyl site new, incoming tRNA
• Once A-site is occupied the peptidyl transferase
  makes the peptide bond, releasing the growing
  peptide chain from the tRNA in the P-site
• mRNA-tRNA-peptide complex shifts to P-site by 3
  nt, requires elongation factors and GTP to move,
  process repeated

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Termination

• Ending of the process
• Stop codon in the A-site which has no AA
  associated with it
• Signals GTP-dependent release factors which will
  cleave the peptide from the tRNA in the P-site
• Complex dissociates

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Polyribosome or Polysome

• Once mRNA has passed thru small subunit and can
  interact with another small subunit and begin a
  2nd initiation complex
• Efficient use of components of protein synthesis
  in a given time
• Occurs in both cell types

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Eukaryotic Transcription is More Complex

• Larger ribosome, more complex
• Longer-lived mRNA several hr vs. min
• Events of transcription
• 5 cap required for efficient translation short
  recognition sequence around AUG 5-ACCAUGG
  called the Kozak sequence, binds the rRNA of
  small subunit
• Doesnt use f-met but does require unique tRNAmet
• Similar protein factors homologous counterpart
  to prokaryotes but usually many more
• Ribosomes in association with ER proteins to be
  secreted or used in the membrane
• tunnel in large subunit may move proteins into
  the ER

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Inborn Errors in Metabolism


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Alkaptonuria

• People cant metabolize alkapton
  2,5-dihydroxyl-phenyl acetic acid (homogentisic
  acid) and it builds up in the cells and excreted
  in the urine
• diapers turn black because of oxidation
• ears and nose darken as HA accumulates in
  cartilage and in joints, benign arthritis
• not serious but persistent and inherited as
  simple recessive trait
• Scientists called it a unit factor and ferments
  (gene and enzyme)
• lack a critical substance

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Phenylketonuria (PKU)

• Different path is blocked
• Results in mental retardation
• Autosomal recessive disease
• Cant convert Phe to Try missing Phe
  hydroxylase 30 active in herterozygous
  individual
• by product ends up in urine and Phe in CSF that
  causes mental retardation
• 1 in 11,000 births, screen to prevent mental
  retardation with early detection restrict diet
  with low Phe levels

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One Gene One Enzyme Theory

• Used Neurospora
• Grow them on complete medium to grow mutant
  strains made with irradiation that cant grow on
  minimal medium
• If growth on minimal medium then not mutation
  related to nutritional needs
• No growth meant nutritional growth mutation
• To determine type of mutation need to look at
  growth on other media

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One Gene One Enzyme

• Found mutations in just about every pathway so
  thought that it was just in the enzymes
• Needed to do biochemical analysis studied 7
  mutants in that didnt make arginine
• Used ornithine, citrulline and arginine in the
  media
• 4 of 7 mutants grew when any of the 3 where
  present
• Arg 2 and 3 grew when citrulline or Arg present
• Arg 1 grew only when Arg provided

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Enzymes Involved

• Arg 4-7 block must be before any of the 3 are
  involved in the path
• Arg 2 and 3 the block is between the making of
  ornithine and citrulline because when only
  ornithine, no growth but citrulline restores
  growth
• Arg 1 the block is between citrulline and Arg
  as the mutant only grows when the end-product of
  the pathway is present

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One Gene One Polypeptide

• Realized that not all mutations occur in enzymes
  study of sickle cell anemia patients
• hemoglobin is not an enzyme
• Alter of one gene one enzyme to one gene one
  polypeptide
• nearly all enzymes are proteins but not all
  proteins are enzymes and knew that genes dictate
  protein expression
• proteins show substructure of 2 or more subunits
  and have quaternary structure

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Sickle Cell Anemia

• Cells under low O2 tension become elongated and
  curved, plugging capillaries, deprive tissue from
  O2 sickle crisis
• Also anemic because the cells are removed bones
  are larger and the heart is more dilated

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Sickle Cell Genes

• 2 genes HbA and HbS
• HbA HbA normal HbS HbS sickle cell disease
  and HbA HbS sickle cell trait (much less
  severe, carriers and pass on to 50 of offspring)
• 1 in 625 African-Americans
• 1 in 145 African-American married couples are
  carriers 25 chance of child having sickle cell
  disease
• HbA and HbS separate differently during
  electrophoresis on starch agar

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Identification of Mutation

• HbA migrates further than HbS
• Used 2-D electrophoresis to find the altered
  amino acid
• Substitution of nucleotide to change the gly to
  val cause of change in shape of protein under
  low O2

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Human Hemoglobins

• Hgb is usually 2 ? chains that are 141 AA long
  and 2 ? chains that are 146 AA long assumes a
  quaternary structure
• Genes for Hgb are developmentally regulated
• most adults are HbA (2 ?/2 ?) 98 of all Hgb
  after 6 months of age, rest are HbA2 ( 2 ?/2 ?,
  similar ?)
• Gower 1 Hgb in embryos 2 ? (similar to ?) and
  2 ? (similar to ?) by 8 weeks it changes to
  another type Hgb F (fetal Hgb) that have 2 ? and
  2 ? chains
• Hgb F persists thru birth and then replaced by
  HgbA - ? is similar to ? - differ by 1 AA

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Protein Structure

• Structure of proteins is basis of incredible
  complexity
• Polypeptides and proteins are both made of AA but
  differ in the state of assembly and functional
  capacity
• polypeptides are precursors to proteins
• polypeptides fold and assume a 3-D structure and
  join with other polypeptide subunits to make a
  fully functional protein

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Polypeptides and AA

• Linear array of AA, similar to nucleotides
• AA starts with a central C that has a carboxyl
  and amino group attached, a single H and an R
  group that makes each AA unique
• R group has 4 main classes
• non-polar, hydrophobic Ala, Val, Leu, Ile, Pro,
  Met, Phe, Trp
• polar, hydrophilic Gly, Ser, Thr, Cys, Tyr,
  Asn, Gln
• positively charged (basic) Lys, His, Arg
• negatively charged (acidic) Glu, Asp
• 200 AA per peptide 20200 many different
  combinations of AA to make proteins
• also have 4 levels of protein structure

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Peptide Bonds

• Amino group of one AA is attached to the carboxyl
  group of another AA in a dehydration rxn
• gt10 AA polypeptide each has a free amino group
  and free carboxyl group at the other end
• directionality

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Primary and Secondary Structure

• 1 linear backbone sequence of the AA that
  will dictate the rest of the structure
• 2 - regular or repeated configuration based on
  closely aligned AA
• ? helix spiral chain of Aa stabilized by
  H-bonds, R group sticks out of the spiral
• ? sheet single polypeptide chain folds back on
  itself to make a pleated sheet may be parallel
  or antiparallel, stabilized by H-bonds

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2 Structures

• Get a mixture of ? helices and ? sheets in
  proteins that are usually globular in nature
• ? sheets core with areas of ? helix so H2O
  soluble
• less H2O soluble and rigid proteins have more ?
  sheets, see in fibroin protein from silk moth

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3 Structure

• Defines 3-D structure of entire chain,
  characteristic for each protein
• 3 important aspects in stabilizing and
  determining 3-D conformation dictates specific
  function of protein
• covalent bonds between 2 cys residues to make
  cystine
• polar, hydrophilic R groups on outside interact
  with H2O
• nonpolar, hydrophobic R groups on inside away
  from H2O
• Dependent on 1 AA relative to all the others

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Quaternary Structure

• Only those with more than one polypeptide chain
  oligomeric
• each chain is called a protomer or subunit
• Hgb is a tetramer of 2 types of polypeptides