From Gene to Protein

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Chapter 17

• From Gene to Protein

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Introduction

• The information content of DNA is in the form of
  specific sequences of nucleotides along the DNA
  strands.
• The DNA inherited by an organism leads to
  specific traits by dictating the synthesis of
  proteins.
• Proteins are the links between genotype and
  phenotype.

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The Connection Between Genes and Proteins

• The relationship between genes and proteins was
  first proposed by ArchibaldGarrod, a British
  physician, in 1909.
• He observed that inherited diseases reflect a
  patient's inability to make a particular enzyme,
  which he referred to as "inborn errors of
  metabolism"

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• He was working with the disorder alkaptonuria, in
  which the urine turns black due to the chemical
  alkapton which darkens with exposure to air.
• Hence, a patient with the disorder alkaptonuria
  produces a dark urine, presumably because these
  people lacked the enzyme found in normal
  individuals who are able to convert the alkapton,
  to another substance.
• SO- there is one gene for each protein. 

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Transcription and Translation Overview.

• Genes provide the instructions for making
  specific proteins.
• The bridge between DNA and protein synthesis is
  RNA.
• Transcription and Translation are the processes
  of going from DNA to RNA to Protein.

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RNA

• RNA is chemically similar to DNA, except that it
  contains ribose as its sugar and substitutes the
  nitrogenous base uracil for thymine.
• An RNA molecule almost always consists of a
  single strand.
• In DNA or RNA, the four nucleotide monomers act
  like the letters of the alphabet to communicate
  information.

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Transcription

• During transcription, a DNA strand provides a
  template for the synthesis of a complementary RNA
  strand.
• Transcription of a gene produces a messenger RNA
  (mRNA) molecule.

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Translation

• During translation, the information contained in
  the order of nucleotides in mRNA is used to
  determine the amino acid sequence of a
  polypeptide.
• Translation occurs at ribosomes.

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The Genetic Code

• In the triplet code, three consecutive bases
  specify an amino acid, creating 43 (64) possible
  code words.
• The genetic instructions for a polypeptide chain
  are written in DNA as a series of
  three-nucleotide words.
• During translation, blocks of three nucleotides,
  codons, are decoded into a sequence of amino
  acids.

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Genetic Code

• To extract the message from the genetic code
  requires specifying the correct starting point.
• This establishes the reading frame and subsequent
  codons are read in groups of three nucleotides.

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Deciphering the Genetic Code

• The task of matching each codon to its amino acid
  counterpart began in the early 1960s.
• Marshall Nirenberg determined the first match
  UUU coded for the amino acid phenylalanine.
• By the mid-1960s the entire code was deciphered.
• 61 of 64 triplets code for amino acids.

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Transcription

• RNA polymerase pries the 2 strands of DNA apart.
• It hooks the RNA nucleotides together as they
  base-pair along the DNA template.
• The growing RNA molecule forms in the 5 ? 3
  direction.

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• A promoter initiates the transcription sequence.
  The promoter is a specific sequence of DNA
  nucleotides.
• A terminator sequence signals the end of
  transcription.
• The length of DNA that is transcribed into an RNA
  molecule is called the transcription unit.

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Promoters

• The promoter serves as the binding site for the
  RNA polymerase and determines which DNA strand is
  the template.
• Certain sections of the promoter are important
  for the RNA polymerase binding.
• In eukaryotes, certain proteins, called
  transcription factors, mediate the binding of RNA
  polymerase and the initiation of transcription.

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• The transcription factors and the RNA polymerase
  bound to the promoter is called a transcription
  initiation complex.
• One crucial promoter DNA sequence is called a
  TATA Box, it contains the nucleotides TATA.
• TATAAAA with its complement
• ATATTTT is one TATA box.

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Termination and Processing

• RNA is transcribed until a DNA terminator
  sequence is transcribed.
• Once the RNA is transcribed, it must be modified.
• Enzymes modify the pre-RNA by altering the ends
  of the RNA molecule.

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• The 5 end is capped off with a modified form of
  Guanine. This 5 cap protects the RNA from
  hydrolytic enzymes and it functions as an attach
  here signal for the ribosomes.
• The 3end is modified by the addition of a poly
  (A) tail which consists of 50-250 Adenines, which
  inhibits degredation of the RNA and helps
  transport the RNA out of the nucleus.

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RNA Splicing

• A third modification that RNA undergoes before
  translation is splicing.
• Sections of noncoding RNA (introns) must be
  removed.
• The remaining exons form the mRNA molecule that
  enters the cytoplasm.

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• Particles called small nuclear ribonucleoproteins
  or snRNPs (snurps) recognize the splice sites.
• These snRNPs join with proteins to form a
  spliceosome which cuts the RNA at the appropriate
  sites and then joins the exons together.

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Translation

• During translation, tranfer RNA or tRNA transfers
  amino acids to the ribosome to begin protein
  assembly.
• Each tRNA links to a particular mRNA in the
  ribosome, and releases its amino acid to form the
  polypeptide chain.
• If mRNA is UUU then the tRNA anticodon is AAA by
  base-pairing rules.

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Transfer RNA

• tRNA is made from the DNA template in the
  nucleus. It travels to the cytoplasm, where
  translation occurs.
• There are about 45 different tRNA molecules that
  carry the 20 amino acids.
• It would appear that there should be 61, but
  wobble, and inosine play a role in this
  phenomenon.

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• Wobble is a relaxation of base pairing rules
  which allows for versatility in the identity of
  the third base in the codon.
• Uracil in the third position of the anticodon can
  base pair with A or G.
• Some tRNAs have a modified third base, inosine
  which can base pair with U, C or A.

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Building a Polypeptide

• Translation occurs in three stages
• Initiation
• Elongation
• Termination

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Initiation

• To initiate translation a 30S ribosomal subunit
  binds to a short nucleotide sequence on the mRNA
  called the ribosome binding site.
• However, translation doesn't usually begin until
  the 30S ribosomal subunit reaches the first AUG
  sequence in the mRNA.

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• For this reason, AUG is known as the start codon.
  At this point, an initiation complex composed of
  the 30S subunit, a tRNA having the anticodon UAC
  and carrying an altered form of the amino acid
  methionine (N-formylmethionine or f-Met), and
  proteins called initiation factors is formed.

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Initiation
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A 50S ribosomal subunit then attaches to the
initiation complex and the initiation factors
leave. This forms the 70S ribosome.
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Elongation

• The joining of individual amino acids to form a
  protein or polypeptide is known as the elongation
  phase of translation.
• There are two sites on the 70S ribosome.
• The A or acceptor or aminoacyl site is where an
  aminoacyl-tRNA first attaches.
• The P or peptide site is where the growing amino
  acid chain is temporarily being held by a tRNA as
  the next codon in the mRNA is being read.

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• During peptide bond formation, the amino acid
  chain or peptide moves from the tRNA at the
  P-site and forms a peptide bond with the new
  amino acid attached to the tRNA at the A-site.
• The peptide bond is formed by a ribozyme, an
  enzyme composed of RNA, called peptidyl
  transferase (def).
• The now uncharged tRNA at the P-site leaves the
  ribosome through an adjacent portion called the
  E-site (exit site) to eventually pick up a new
  amino acid and be recycled.
• Meanwhile, the 70S ribosome moves a distance of
  one codon down the mRNA through a process called
  translocation to allow decoding of the next codon
  in the message.

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Elongation
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Termination

• This process continues over and over again in the
  5' to 3' direction until the ribosome hits a stop
  codon.
• A stop codon is a series of three mRNA bases
  coding for no amino acid and thus terminates the
  protein chain.
• UAA, UAG, UGA are the three stop codons in the
  genetic code.

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• Stop codons do not code for an amino acid because
  they cannot be recognized by a tRNA.
• Proteins called release factors free the protein
  from the tRNA and the two ribosomal subunits come
  apart to be recycled.
• During this elongation process, the protein has
  assumed its three-dimensional functional shape.

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Termination
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Translation Animation
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Ribosomes

• Ribosomes facilitate the coupling of tRNA
  anticodons with mRNA codons during protein
  sythesis.
• The ribosome is made of 2 subunits which are
  constructed of ribosomal RNA.
• Ribosomes have 3 binding sites for tRNA.

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Ribosomal Binding Sites

• The P site holds the tRNA carrying the growing
  polypeptide chain.
• The A site holds the tRNA carrying the next amino
  acid.
• The E site is the exit site or the place where
  the tRNA is discharged.

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Mutations

• Point mutations are changes in one base pair of a
  gene.
• Substitutions are the substitution of one
  nucleotide and its complement with another pair
  of nucleotides.
• Missense mutations are when the altered codon
  still codes for the correct amino acid (it makes
  sense).
• Nonsense mutations occur when the codon is
  changed to stop codon.

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• Insertions and Deletions are when a nucleotide
  pair is added or lost from the original sequence.
  
• If the nucleotides added or deleted are not in a
  multiple of 3 then a frameshift mutation will
  occur.

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Mutagens

• Mutations can occur as a result of errors during
  DNA replication, repair or recombination. These
  are spontaneous mutations.
• Physical and chemical agents can also cause
  mutations high energy radiation is one such
  agent.