NUCLEIC ACIDS AND PROTEIN SYNTHESIS

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CHAPTER 21

• NUCLEIC ACIDS AND PROTEIN SYNTHESIS

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What Role Do Nucleic Acids Play?

• DNA
• Contained in cell nucleus
• All information needed for the development of a
  complete living system
• Every time a cell divides, cells DNA is copied
  and passed to the new cells.
• RNA
• Part of the process of making proteins from
  genetic information encoded in DNA
• RNA transcribes the information contained in the
  genes and carries the code out to the
  protein-making machinery

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A. Components of Nucleic Acids

• DNA and RNA are both nucleic acids
• Both unbranched polymers of repeating
  nucleotide monomers
• Each nucleotide has three components a
  nitrogenous base, a five-carbon sugar, and a
  phosphate group.
• Nitrogen-containing bases
• Derivatives of pyrimidine or purine.
• Adenine (A) and guanine (G) are purines, and
  cytosine C and thymine (T) are pyrimidines. RNA
  uses the same bases, except that T is replaced by
  uracil (U).
• Nitrogenous Base Structures

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The Four DNA Bases, and Uracil

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Ribose/Deoxyribose

• Both RNA and DNA contain 5-carbon sugars.
• RNA ribose
• DNA deoxyribose
• The carbons in the sugars are numbered with
  primes.

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Nucleosides/Nucleotides

• Nucleoside base sugar
• Nucleotide base sugar phosphate group
• Tide contains phosphates!
• Naming Adenine ribose adenosine
• Adenine deoxyribose deoxyadenosine
• Naming nucleosides of the other bases follows
  the same pattern.
• Naming nucleotides nucleoside name followed by
• 5-monophosphate
• ex. Adenosine 5-monophosphate (AMP) or
  deoxyadenosine 5-monophosphate (dAMP)

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Nucleosides/Nucleotides
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Nucleoside Di- and Triphosphates

• Any nucleoside 5-monophosphate can bind
  additional phosphate groups, forming a
  diphosphate or triphosphate
• For example, you can form the famous ADP
  (adenosine 5-diphosphate) and ATP (adenosine
  5-triphosphate) through the addition of
  phosphate groups.
• The same can be done for nucleosides of other
  bases (ex. GTP, CDP, etc)

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Nucleoside Di- and Triphosphates
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Lets practice

• Identify each of the following as a nucleoside or
  a nucleotide
• Guanosine
• Nucleoside -- phosphate not part of the name
• Deoxythymidine
• Nucleoside
• Cytidine 5-monophosphate
• Nucleotide -- phosphate is part of the name

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B. Primary Structure of Nucleic Acids

• The nucleotides are linked together from the 3
  -OH of the sugar in one nucleotide to the
  phosphate on the 5 carbon of the next
  nucleotide.
• This phosphate link is called a phosphodiester
  bond. The chain formed from multiple
  phosphodiester bonds forms the backbone of a
  strand of DNA.
• Phosphodiester bond formation
• Sequence of bases in the nucleic acid primary
  structure. The sequence is written with 5 and
  3 ends labeled, for instance -- 5-ACGT-3

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A Single Strand of RNA (ACGU)

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C. DNA Double Helix

• In the 1940s, it was discovered that the percent
  of A in an organism T. Likewise, C G.
• What might this suggest?
• Base pairing rules in two complementary strands
  of DNA, A always base pairs with T, and C always
  base pairs with G.
• 1953 DNA discovered to be a double helix (winds
  like a spiral staircase)
• DNA Double Helix
• The strands are antiparallel.

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A DNA Molecule (at least according to the
computer)

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D. DNA Replication

• Whenever cells divide, the DNA in the cells needs
  to replicate -- an exact copy of the DNA needs to
  be passed to the new cells.
• Replication begins when the enzyme helicase
  unwinds a portion of the helix by breaking
  hydrogen bonds between the strands.
• A nucleoside triphosphate bonds to the sugar at
  the end of the growing new strand. Two phosphate
  groups are cleaved (this provides the energy for
  the reaction)
• And DNA polymerase catalyzes the formation of the
  new phosphodiester bond.

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DNA Replication

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DNA Replication cont.

• When the entire DNA double helix has been
  replicated, one strand will be from the original
  DNA and one will be a newly synthesized strand.
• This is why the process is called
  semi-conservative replication
• Ensures an exact copy of the original DNA through
  base pairing rules
• The process of replication has directionality.
  New nucleotides are only added onto the 3 end of
  a growing chain.
• The chain that grows in the 5 --gt 3 direction
  leading strand. Continuously synthesized.
• The chain that grows in the 3 --gt 5 direction
  lagging strand.

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How is the lagging strand synthesized?

• As replication forks (bubbles along the double
  helix) open up, short fragments of the lagging
  strand are synthesized in the 5 --gt 3 direction
  as space allows. These fragments are called
  Okasaki fragments.
• These fragments are eventually joined by DNA
  ligase to create a continuous strand of DNA.

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Synthesis of Lagging Strand

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E. RNA and Transcription

• RNA is similar to DNA, except
• Different sugar (ribose instead of deoxyribose)
• The nitrogen base uracil replaces thymine
• RNA molecules are single stranded (not double
  stranded)
• RNA molecules are much smaller than DNA molecules

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Three Types of RNA

• Ribosomal RNA (rRNA) -- contained in ribosomes,
  the site of protein synthesis
• Messenger RNA (mRNA)
• Carries genetic info from DNA in nucleus to
  ribosomes in cytoplasm for protein synthesis
• Is a copy of the gene
• Transfer RNA (tRNA) -- brings the appropriate
  amino acid to the ribosome during the process of
  protein synthesis. Each tRNA contains an
  anticodon (three bases complementing a three-base
  segment on the mRNA) which allows for match-up
  with exact amino acid.

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Transcription Synthesis of mRNA

• Begins with unwinding of a section of the DNA
  containing the gene needing to be copied
• Initiation point (signal) for transcription
  TATAAA
• RNA polymerase moves along the template strand in
  the 3 to 5 direction, allowing it to synthesize
  RNA adding new nucleotides to the 3 end of the
  new strand.
• When a termination signal is reached, the mRNA is
  released, and DNA recoils back into its double
  helix structure.

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Transcription

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Processing of mRNA

• Happens in eukaryotic cells, but not in
  prokaryotes
• Eukaryotic genes contain introns -- sections that
  do not code for protein -- interspersed with
  coding sections called exons
• Prokaryotic genes do not contain exons and
  introns
• Prior to leaving the nucleus, the eukaryotic mRNA
  undergoes processing -- introns get snipped out,
  or spliced.

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mRNA Processing

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Regulation of Transcription

• The cell goes not make mRNA randomly. There are
  certain proteins which are constantly needed, but
  not very many.
• Most mRNA is synthesized in response to cellular
  needs for a particular protein. Regulation is at
  the level of transcription.
• Prokaryotic cells regulate transcription by means
  of the operon -- more than one gene under the
  control of the same regulatory center.
• Control site promoter (place where RNA
  polymerase binds) and operator (place where
  repressor may or may not bind)

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The lac operon (prokaryotes)

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F. The Genetic Code Codons

• A sequence of three bases is called a codon.
• Each codon specifies an amino acid in the
  protein.
• All 20 amino acids have their own codon -- some
  amino acids have more than one.
• Three codons specify the stop of protein
  synthesis -- they are UAG, UGA, and UAA.
• AUG signals the start of protein synthesis and
  also encondes the amino acid methionine.

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The Genetic Code
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G. Protein Synthesis Translation

• Occurs at ribosomes, outside of nucleus
• tRNA are used to translate each codon into an
  amino acid
• Anticodon in the bottom loop is a three-base
  complement to the codon in the mRNA
• Amino acid is attached to the stem on the
  opposite end of the tRNA via an aminoacyl-tRNA
  synthetase..

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A Single tRNA

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Initiation of Protein Synthesis

• Both ribosomal subunits and an mRNA combine,
  recognizing the start codon on the mRNA
• The appropriate tRNA binds to the codon
• Next, the appropriate tRNA binds to the second
  codon on the mRNA a peptide bond is formed
  between the two neighboring amino acids.
• The first tRNA dissociates
• The ribosome shifts down the mRNA chain, allowing
  space for the next tRNA down the line to float in
  and bind
• This process continues until a stop codon is
  reached.

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Termination of Protein Synthesis

• When the ribosome reaches a stop codon, protein
  synthesis ends.
• The entire complex dissociates, and the peptide
  is released. The peptide can fold.

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Translation Overview

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H. Genetic Mutations

• Mutation change in DNA sequence, altering the
  amino acid sequence as well
• Causes of mutation radiation (X rays/UV light),
  chemicals called mutagens, perhaps viruses
• Mutation in somatic cell body cells resulting
  from division contain the mutation
• Could lead to tumor/cancer
• Mutation in germ cell (egg or sperm) offspring
  will contain mutation
• Mutations can affect function of important enzymes

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Types of Mutations

• Replacement of one base with another
  substitution mutation
• May or may not change the individual amino acid,
  but no downstream effect
• Frameshift mutation base is added to, or
  deleted from, the sequence. Changes reading
  frame.
• The amino acid in question is affected, as well
  as all downstream amino acids (out of frame)

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Types

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Effect of Mutations

• If an enzyme, may completely lose activity
• Does the mutation change the active site
  directly?
• If not, does it alter the 3D shape of the protein
  enough so that the substrate can no longer bind?
• A defective protein (due to mutation) may result
  in genetic disease.

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Practice

• For the following mRNA sequence
• 5-ACA-UCA-CGG-GUA-3
• If a mutation changes UCA to ACA, what happens to
  the protein?
• What happens if the first U is removed from the
  sequence?

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Genetic Diseases

• Result of a defective enzyme, resulting from a
  mutation
• Example -- albinism
• An enzyme normally converts tyrosine to melanin
  (pigment causing hair/skin color)
• If this enzyme is defective, no melanin produced
  albinism

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J. Recombinant DNA

• Cutting and pasting DNA from the same organism,
  or from different organisms
• The resulting DNA is called recombinant
• Has allowed for the production of human insulin,
  interferon, human growth hormone

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Preparing Recombinant DNA

• Using E. coli (prokaryotic) as an example some
  bacteria contain circular DNA called plasmids.
• Plasma membranes are dissolved and plasmid DNA
  isolated
• A restriction enzyme (recognizes a certain DNA
  sequence and cuts) cuts through the plasmid
• Another piece of DNA can be placed into the cut
  plasmid, and ends sealed
• The recombinant plasmids can be placed into cells

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Recombinant DNA Prep

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The Point of Recombinant DNA

• If you have a cell containing your recombinant
  plasmid when the cell multiplies, each new cell
  will contain this plasmid
• If your recombinant plasmid contains a gene
  (protein) of interest following a promoter, you
  can stimulate the cells to make large amounts of
  your protein of interest

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Polymerase Chain Reaction

• If you only have one copy (or a few copies) of
  one gene, this is a method to amplify (make a lot
  of copies) the gene quickly.
• Three steps
• Heat your DNA of interest -- the double strands
  will separate
• Primers (short sequence complementary to each
  end) are added -- they anneal to the end of your
  single strands
• The addition of DNA polymerase and free
  nucleotides extends along the single strand,
  filling in until each double strand is complete.

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PCR

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K. Viruses

• Cannot replicate without a host cell
• Invades the host cell, taking over materials
  necessary for protein synthesis and growth
• Viral infection
• Virus inserts its genetic material (DNA or RNA)
  into host cell
• Material is replicated into DNA form
• The viral DNA is used to make viral proteins via
  transcription and translation
• In some cases, the host cell will lyse, releasing
  new viral particles

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Life Cycle of a (Lytic) Virus

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Reverse Transcription

• Viruses that use RNA as their genetic material
  must make viral DNA once inside the host cell
• It does so via the enzyme reverse transcriptase.
  
• A virus which contains RNA and uses this process
  is called a retrovirus.

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AIDS/HIV A Retrovirus

• HIV destroys helper T cells (important in the
  immune response)
• Thus, AIDS is defined by opportunistic infections
• Treatments for AIDS?
• Nucleoside analogs transcription enzymes put
  false nucleotides into strands, proteins cant be
  made
• Protease inhibitors HIV protease chops the
  final viral peptide into useable form. If
  protease blocked, viral proteins are nonfunctional