From Gene to Protein

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From Gene to Protein
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Proteins are the link between genotype and
phenotype.

• The DNA inherited by an organism leads to
  specific traits by dictating the synthesis of
  proteins.
• Gene expression involves 2 stages
• Transcription
• Translation

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Genes Specify Proteins via Transcription and
Translation

• 1909 Archibald Garrod (Great Britain)
• Hypothesized that genes dictate phenotype
• ex Black Urine (alkaptonuria)
• black because it lacks the gene required to
  break down the enzyme alkapton, which darkens
  with air exposure.
• 1930s George Beadle Boris Ephrussi speculated
  various mutations caused eye color in fruit
  flies, by preventing production of an enzyme
  catalyzing the step.

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More Studies

• Beadle Edward Tatum began working w/bread mold
  (Neurospora crassa). They bombarded it w/x-rays,
  then looked among the survivors for mutants that
  differed in their nutritional needs from the wild
  type. (Figure 17.2)
• Discovered one gene per mutation. Came up w/ the
  one gene one polypeptide hypothesis
• Some genes code for RNA molecules that have
  important functions w/in the cell even though
  they are never translated into protein.

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Genes provide instruction for making specific
proteins

• Since genes dont directly make the proteins, we
  rely on another set nucleotides, RNA, outside the
  nucleus.
• DNA RNA
• TACG UACG
• Double Helix Single Strand
• Deoxyribose Ribose
• Heredity Protein Synthesis
• 1 Type unique to 3-Types
• each individual mRNA, rRNA, tRNA

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Genes are hundreds to thousands nucleotides long.

• The primary structure of a polypeptide is made up
  of only 20 amino acids.
• Getting from DNA to protein requires 2 steps
• transcription
• translation

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Transcription

• DNA provides a template for synthesis of a new
  RNA strand.
• This transcript is the recipe for making a
  specific protein.
• This RNA molecule is called messenger RNA (mRNA),
  because it carries the genetic message from DNA
  to the protein synthesizing components of the
  cell.

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DNA to RNA to Protein

• The transcription of a protein-coding eukaryotic
  gene results in a pre-mRNA, and RNA processing
  yields the finished mRNA.
• The initial RNA transcript from any gene,
  (including those coded for RNA that is not
  translated into protein) is called a primary
  transcript.

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Translation

• The actual synthesis of the polypeptide under the
  direction of the mRNA molecule.
• Sort of a change in language.
• This happens at a site outside the nucleus in the
  cytoplasm on ribosomes that link the amino acids
  into polypeptide chains - rRNA.
• Each mRNA transcript can be translated many
  times.
• tRNA builds the polypeptide chain by bring
  anticodons to match codon to build chain

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The Genetic Code

• 4 nucleotides to make 20 amino acids
• Triplets of nucleotide bases code for all the
  amino acids. 43 64 not 20. Triplet Code
• There is more than one codon for several of the
  20 amino acids, including start stop codons.
• During transcription only one side of the DNA
  strands is transcribed, template strand.
• The mRNA strand is a complementary strand not
  identical to DNA, and follows Chargoffs rules.

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Amino Acids

• Each amino acid consists of an alpha carbon atom
  to which is attached
• a hydrogen atom
• an amino group (hence "amino" acid)
• a carboxyl group (-COOH). This gives up a proton
  and is thus an acid (hence amino "acid")
• one of 20 different "R" groups. It is the
  structure of the R group that determines which of
  the 20 it is and its special properties.
• The amino acid shown here is Alanine.

Amino acids are the building blocks (monomers)
of proteins. 20 different amino acids are used
to synthesize proteins. The shape and other
properties of each protein is dictated by the
precise sequence of amino acids in it.
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The Amino AcidsFor each amino acid, both the
three-letter and single-letter codes are given.
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essential amino acids

• The Essential Amino Acids
• Histidine
• Isoleucine
• Leucine
• Lysine
• Methionine (and/or cysteine)
• Phenylalanine (and/or tyrosine)
• Threonine
• Tryptophan
• Valine

• Humans must include adequate amounts of 9
  amino acids in their diet.
• These "essential" amino acids cannot be
  synthesized from other precursors.
• However, cysteine can partially meet the need
  for methionine (they both contain sulfur), and
  tyrosine can partially substitute for
  phenylalanine.

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Codons

• RNA molecules synthesizes in an antiparallel
  direction to the template strand of DNA
• DNA runs 3 to 5, so RNA will run 5 to 3
• The non template stand matches the mRNA strand,
  except T is for U, thus sometimes it is referred
  to as the coding strand.
• The mRNA base triplets are called codons. Each
  codon specifies an amino acid, which will take
  its corresponding place in the polypeptide.

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The code

• All triplets code for an amino acid except three,
  the stop codons UAA, UAG, UGA
• AUG codes for methionine (Met) and also the start
  codon
• Genetic messages begin with the mRNA codon, AUG,
  which signals the protein-synthesizing machinery
  to begin translating the mRNA at that location
• There is a redundancy, GAA GAG both code for
  glutamic acid, but neither codes any other amino
  acid (ambiguity)

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More Code

• Reading Frame molecular language
• Read from 5 to 3
• Never overlaps
• Nearly universal for bacteria to humans
• Exceptions include translation systems where few
  codons differ slightly from standard ones in a
  few eukaryotic single-celled organisms.
• For the most part the code is universal has
  existed from the beginning of life.

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Transcriptions 3 StagesInitiation, Elongation,
Termination

• Initiation occurs when an RNA polymerase
  identifies the beginning of a protein chain on
  DNA, binds to it, and unwinds and "unzips" the
  DNA to create a template for RNA to be built.
• In elongation, the polymerase synthesizes RNA
  along the length of the DNA template. Errors are
  proofread for and can be edited out during this
  stage.
• Termination is the end of the RNA creation, and
  is usually signaled by a palindromic sequence in
  the nucleotides that causes a physical loop the
  RNA cannot bind to it's a little like folding
  back a bit of tape to create a blunt nonsticky
  end.
• Prokaryotes RNA can be immediately usable as
  mRNA
• Eukaryotes must first undergo processing

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Initiation, Elongation, Termination
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Transcription Vocabulary

• RNA Polymerase Enzyme that links together
  growing chain of ribonucleotides
• Promoter Specific nucleotide sequence in DNA
  that binds RNA polymerase indicates where to
  start transcription
• Terminator In prokaryotes, as special sequence
  of nucleotides in DNA that marks the end of a
  gene. It signals RNA polymerase to release newly
  made RNA molecule, which then departs from the
  gene.
• Transcription Unit A region of DNA molecule that
  is transcribed into a RNA molecule.

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The Promoter of the Gene

• Transcription point
• Actual nucleotide plus several dozen nucleotide
  pairs upstream from start point
• Binding site for RNA Polymerase
• Determines which two strands to copy
• In prokaryotes RNA Polymerase specifically
  recognizes and binds to promoter
• In Eukaryotes a collection of proteins call
  transcription factors mediate the binding of RNA
  Polymerase and the initiation of transcription.

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TATA Box

• The TATA box can be found in various species
  ranging from simple eukaryotes such as baker's
  yeast to more complex organisms such as humans.
  The TATA box assists in directing RNA polymerase
  II to the initiation site downstream on DNA.

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Elongation

• RNA polymerase moves along the DNA , it continues
  to untwist the double helix, exposing 10 20
  nucleotides at a time to pair with the RNA
  nucleotides
• Enzymes add nucleotides to the 3 end of the
  growing RNA
• RNA molecule peels away, double helix reforms
• 60 nucleotides per second
• Single gene can be transcribed simultaneously be
  several molecules of RNA polymerase, following
  each other like a convoy.

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Termination

• Differs in prokaryotes eukaryotes
• In prokaryotes transcription proceeds through the
  terminator sequence of DNA, the terminator
  signals the end, causing polymerase to detach
• In eukaryotes, the pre-mRNA
• (AAUAAA polyadenylation signal) is cleaved
  from the growing RNA chain while RNA polymerase
  II continues to transcribe DNA
• The polymerase continues transcribing 100s of
  nucleotides past the site of pre-mRNA was
  released. Not sure why or how it then just falls
  off

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Eukaryotic Cells Modify RNA after Transcription

• Both ends of the primary transcript are altered
• The 5 end (transcribed 1st) is capped off w/a
  form of guanine after 1st 20-30 nucleotides
  called the 5 cap
• AAUAAA forming poly A tail while still in nucleus
  for 3 end
• Certain molecules are cut out, w/remaining
  spliced back together

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WHY?

• They facilitate the export of the mature mRNA
  from the nucleus
• They help protect mRNA from degradation by
  hydrolytic enzymes.
• Once the mRNA reaches the cytoplasm, both
  structures help ribosomes attach to the 5 end of
  the mRNA
• See fig. 17.9, pg 317 Campbell/Reese 7th ed.

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Split Genes and RNA Splicing

• Eukaryotic nucleus removes a large portion of
  initial RNA molecule
• Cut Paste job RNA splicing
• Ave. length of transcription unit is about 8000
  nucleotides
• It takes only 1200 nucleotides to code for
  average sized protein of 400 amino acids
• In other words, the sequence of DNA nucleotides
  that code for an eukaryotic polypeptide is
  usually not continuous.

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INTRONS EXONS

• Introns The noncoding segments of nucleic acid
  that lie between the coding regions
• Exons The coding regions that are eventually
  expressed
• Terms apply to both DNA RNA
• In making a primary transcript from a gene, RNA
  polymerase II transcribes both introns and exons
• from the DNA, but mRNA enters the cytoplasm
  abridged
• Introns cut out and exons spliced together

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How?????

• The signal for splicing is a short nucleotide
  sequence at each end of the intron
• Small nuclear ribonucleoproteins (snRNPs)
  recognize these splice sites
• snRNPs join up w/ other proteins to form
  spliceosome, ( slightly smaller than ribosome)
  which interacts w/ certain sites along the
  intron, releasing the intron.
• snRNPs are probably catalytic as well

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Ribozymes

• RNA molecules that function as an enzyme
• Specific base pairing along chain can bind into
  another molecule
• So now we know all catalysts are not only
  protein, but certain organisms RNA can do the
  work of several proteins instead

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Importance of Introns?

• Regulatory role in the cell
• Some introns contain sequences that control gene
  activity in some way
• Complications w/ introns exons means one gene
  can code for more than one polypeptide, depending
  on which segments are treated as exons
  (Alternative Gene Splicing)
• Sex differences in fruit flies are due to
  differences in how male females splice the RNA
  transcribed from certain genes.
• Could be reason why more complex organisms dont
  need as many genes

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Domains

• Proteins often have a modular architecture
  consisting of discrete structural and functional
  regions.
• Exons code for different domains (active sites)
  of a protein Exon Shuffling
• Sometimes contributes to beneficial crossing over
  by providing more terrain

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Translation is the RNA-directed Synthesis of a
Polypeptide

• The message of the transcript of the series of
  codons along mRNA is interpreted in the process
  of translation. It build the polypeptide
  accordingly
• Transfers amino acids from cytoplasmic pool to a
  ribosome. The ribosome adds the each amino acid
  brought to it by tRNA to a growing polypeptide
  chain.
• tRNA translates specific code fro specific amino
  acid

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Anticodons

• Nucleotide triplet of tRNA
• Pairs w/ hydrogen bonding
• Like other RNA molecules tRNA is coded from DNA
  templates, leaves nucleus to exist in cytoplasm
• Each tRNA molecule is used repeatedly in both Pro
  Eukaryotic organisms

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Translation
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Ribosomes
See figures 17.16 17.19 pages 322-25
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Page 327
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Prokaryotes vs Eukaryotes

• Similar transcription/translation method
• Polymerases are different
• Eukaryote transcription factors
• Transcription terminated differently
• Ribosomes slightly different
• Eukaryotic compartmentalization
• eukaryotic cells have complicated mechanisms for
  targeting proteins to the appropriate cellular
  compartment (organelle)

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Point Mutations

• Mutations are changes in genetic material of a
  cell or virus.
• Not to be confused w/chromosomal rearrangements.
• Can effect both protein structure and function.
• Point Mutations
• chemical changes in just one base pair of a gene.

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Point Mutations

• If they occur in a gamete or a cell that gives
  rise to a gamete, then mutation can be
  inheretible.
• Sickle-cell Anemia is an example
• Two categories
• Base pair
• substitutions
• 2. Base pair
• Insertions or Deletions

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Substitution Mutations

• Usually missense mutations altered codon still
  codes for amino acid and thus makes sense,
  although maybe not the right sense.
• A point mutation can also change a codon for an
  amino acid into a stop codon. This is called a
  nonsense mutation. Causes translation to stop
  abruptly. Lead to nonfunctional proteins

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Insertions Deletions

• Additions or losses of nucleotide pairs in a gene
• Disastrous effect
• Alters hole reading frame
• Frameshift Mutations

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Mutagens

• Error during DNA replication, repair, or
  recombination can lead to base-pair
  substitutions, insertions, or deletions as well
  as, mutations affecting longer stretches of DNA.
  (SPONTANEOUS MUTATIONS)
• Physical Chemical agents interact to cause
  mutations in DNA
• Mutagenic radiation, physical like UV light

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Chemical Mutagens

• Several Catagories
• Base Analogs
• DNA replication interference, bonding
• Chemical changes in bases
• Cancer causing agents are mutagenic

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