Genes to Proteins

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Genes to Proteins

• Core 218
• Spring 2007
• Lecture 7

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Criteria for Genetic Material

• The material must be able to replicate.
• The material (genotype)  must control the
  expression of traits (phenotype).
• The material must be able to change in a
  controlled way.

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DNA Replication
Leading Strand
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RNA Primer
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Lagging Strand
Okazaki Fragments
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The Chromosome

• The chromosome (all living things) is composed of
  double stranded deoxyribonucleic acid (DNA)
• DNA is two strands of nucleotides
  (phosphate-ribose-base) that are complementary to
  each other (Chargaffs Rules AT, GC).
• Diagrammatically, DNA can be denoted as a string
  of As, Cs, G, and Ts.
• 5-ATTATAATACGATTCATC-3

• 3-TAATATTATGCTAAGTAG-5
• Adding the complementary strand makes the DNA
  double stranded and satisfies Chargaffs Rules.
• Eukaryotic chromosomes have telomeres - TTAGGG

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Structure of DNA

• 5-ATTATAATACGATTCATC-3
• 3-TAATATTATGCTAAGTAG-5
• (5)P-dR-P-dR-P-dR-OH(3)
• A T T
• T A A
• (3)0H-dR-P-dR-P-dR-P(5)

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Genotype Controls Phenotype

• 1902 Archibald Garrod
• Observation
• Alkaptonuria disease black urine when exposed
  to air (homogentisic acid)
• Alkaptonuria recurs in families appearing in
  siblings but not in parents.
• Hypotheses
• Alkaptonurics lack an enzyme that break down
  alkaptans to normal excretion products.
• Disorder was controlled by a single inherited
  factor
• A genetic disease is caused by a change in the
  ancestors genetic material
• Ignored until Beadle and Tatum

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Genotype Controls Phenotype

• George Beadle (geneticist), Edward Tatum
  (bacteriologist, biochemist).
• One Gene-One Enzyme Hypothesis
• 1940s
• Mutated a fungus Neurospora crassa with X-rays
  and UV light
• Found mutants (auxotrophs)
• Mutations passed on to the next generation

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Beadle and Tatum

• Direct
• Mutations are traceable via the phenotype
• Mutations are inherited from one generation to
  the next
• X-rays and UV light cause mutation in the DNA
• Indirect
• DNA within genes produce enzymes

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Further interpretation

• But as we know today, genes also produce proteins
  with other functions, and also peptides that join
  with other peptides to form a composite protein
  (e.g. hemoglobin)
• Thus we should really say One Gene One
  Peptide
• Beadle, Tatum received the 1958 Nobel Prize for
  Physiology and Medicine

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Joshua Lederberg and Edward Tatum

• Mixed two different auxotrophic strains
  (ABC-D-E-, and A-B-CDE) of bacteria with
  complementary mutations.
• Spread on minimal media.
• Found strains that were now ABCDE

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Gene to Proteins

• Remember One Gene One Enzyme (Beadle and
  Tatum
• The Central Dogma of Molecular Biology

DNA
Transcription
RNA
Translation
Protein
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The Central Dogma of Molecular Biology

• Advantages
• The DNA can retain integrity
• The RNA step allows amplification
• Multiple steps allow multiple points of control

Protein
DNA
Translation
RNA
Transcription
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The Only Violation of the Central Dogma - Prions

• Prions (proteinaceous infectious particle) are
  peptides that are about 250 amino acids in size
• Heat resistant
• Hereditary in 15 of cases (PRNP homozygotes)
• Gene product PrP (Prion protein), common
  alpha-helical form, or rare beta-sheet form
  (scrapei, C-J Disease, Mad Cow)
• Can acquire through infection (i.e., consumption,
  surgery)
• Causes Bovine Spongiform Encephalopathy (BSE, Mad
  Cow Disease)
• Creutzfeldt-Jakob (pronounced Kroytzfelt-
  Yakobe) disease in humans

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Microscopically, one finds vacuolization in
neuronal cytoplasm and dendrites, which gives the
neuropil a spongy appearance

• Gary Baumbach, M.D., Department of Pathology,
  University of Iowa College of Medicine

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Proteinaceous Infections Particles - Prions

• Prions are abnormal proteins that resist protease
• Appear to cause (on contact) normal proteins to
  also fold abnormally
• This results in clumping of the abnormal proteins
• This leads to vacuolization of the brain tissue

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What is a Protein?

• Amino acid
• the basic building block
• All amino acids have a common chemical structure
• A tetrahedral carbon atom
• Four asymmetric groups
• An amino group (NH2)
• A carboxy group (COOH)
• An H atom
• A chemical group (denoted by R) which varies from
  one amino acid to another

In humans, more than 50,000 different proteins
and 20,000 different enzymes are produced
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Amino Acids

• There are 20 amino acids plus 4 slightly unusual
  ones (e.g. creatine)
• 5 Hydrophilic
• Basic lysine, arginine, histidine
• Acidic aspartic acid, glutamic acid
• Uncharged polar
• Nonpolar side chains

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Did You Know?

• Nutrasweet aspartame l-aspartic acid and the
  methyl ester of phenylalanine (dipeptide)
• Methyl group on the phenylalanine can dissociate
  at 86 F, releasing methanol
• Liver converts methanol into formaldehyde, which
  is a known carcinogen
• Equal mostly glucose with a little Nutrasweet
  added plus some filler

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Proteins

• Peptide - Two or more amino acids covalently
  linked by an amide bond between the carboxylic
  acid group of one and the alpha-amino group of
  the other
• Oligopeptide when there are a few aa linked
• Polypeptide when there are many aa linked
  (50-2000 aa chain)

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Proteins

• Proteins a large macromolecule composed of one
  or more polypeptide chains
• Between 5500 and 220,000 D (Daltons)
• 1 Dalton mass of 1 hydrogen atom

The most important thing you need to know is how
to answer the following question What is a
protein?
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Sanger, 1953

• Proteins have unique amino acid sequences
• All amino acids in mammalian proteins are in the
  S-configuration
• Peptide bond is an amide bond
• Amino acids have alpha amino groups and alpha
  carboxyl groups
• Major contribution sequencing proteins

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Elucidation of the Genetic Code

• Ability to sequence polypeptides key to Francis
  Cricks subsequent studies
• 1961, Crick and Gamov
• hypothesized three base code for amino acids
• mutated nucleotide sequences and looked at
  peptide sequence

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The Genetic Code
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What is a Gene?

• A series of nucleotides that has at least
• Initiation or start site, promoter site
• Coding region
• Termination site
• Mimimum size for a gene 1000 base pairs (bp)
• Eukaryotic genes can be as large as 10,000 bp
• Introns non-coding regions, usually splice out
  while processing RNA
• Exons coding regions

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Gene Initiation Sequences

• Upstream
• Prokaryotic -35 region TTGACA, -10 region TATAAT
• Eukaryotic -75 CAT box GGCCAATCT, -30 TATA box
  TATAAA
• Anywhere enhancers

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RNA Polymerase binds to the start 3 antisense
and makes a sense copy. Falls off at the end of
the gene.
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Eucaryotic mRNA processing
Transcription is the process by which a
messenger RNA molecule is synthesized from a DNA
template strand.
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The Spliceosome

• The spliceosome is an assembly of proteins and
  extra DNA in eukaryotes that results in the
  formation of an intact functional gene and is
  part of eukaryotic transcription.
• It is important in that it
• Allows gene rearrangement
• An additional level of regulation
• http//www.maxanim.com/genetics/Spliceosome/Splice
  osome.htm

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Prokaryote Transcription

• What you need to know.
• Transcription is the process by which a
  single-stranded RNA molecule is made from a DNA
  template.
• RNA can be mRNA, tRNA or rRNA.
• The RNA is synthesized by an RNA polymerase which
  is actually a complex molecule composed of
  several different protein subunits.
• There are specific sequences that serve as
  starting or initiation sites for the RNA
  polymerase.
• RNA is made until a termination signal is
  reached.
• http//www.lsic.ucla.edu/ls3/tutorials/gene_expres
  sion.html

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Three Main RNAs in Making Proteins

• Messenger RNA (mRNA)
• Transfer RNA (tRNA)
• Ribosomal RNA (rRNA)
• Ribosomes
• Roughly 2/3 RNA, 1/3 protein
• Bacterial ribosomes are smaller than eukaryotic
  ribosomes
• Two (large and small subunits)

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Translation
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Translation

• Translation is the process by which a peptide
  or protein is synthesized by ribosomal RNA (rRNA)
  from an mRNA template.
• Translation occurs in the cytoplasm.
• http//www.maxanim.com/genetics/Protein20Synthesi
  s/Protein20Synthesis.htm

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http//www.accessexcellence.org/AB/GG/protein_synt
hesis.html