The Blueprint of Life, From DNA to Protein

1
The Blueprint of Life,From DNA to Protein

• Chapter 7

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The Blueprint of Life

• Characteristics of each cell dictated by
  information contained on DNA
• DNA holds master blueprint
• All cell structures and processes directed by DNA

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Overview

• Complete set of genetic information referred to
  as genome
• Genome of all cells is composed of DNA
• Some viruses have RNA genome
• Functional unit of genome is the gene
• Gene codes for gene product
• Gene product is most commonly protein
• Study of transfer of genes is genetics
• Study of sequence of DNA is genomic

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Overview

• Living cells must accomplish two general tasks to
  multiply
• DNA replication
• DNA expression (gene expression)
• Expression involves two process
• Transcription
• Copies information in DNA to RNA
• Translation
• Interpret RNA to synthesize protein
• Flow of information from DNA to RNA to protein
• Central dogma of molecular biology

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Overview

• Characteristics of DNA
• Made up of deoxy-ribonucleotides
• Nucleotides include
• Phosphate group
• 5 carbon sugar
• Deoxyribose
• Nucleotides bond covalently between the 5PO4 of
  one nucleotide and the 3OH of another
• Joining of nucleotides creates an alternating
  sugar-phosphate backbone

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Overview

• Characteristics of DNA
• Each sugar (deoxyribose) molecule is connected to
  a nitrogenous base
• Nitrogenous bases
• Adenine (A) - purine
• Thymine (T) - pyrimidine
• Guanine (G) - purine
• Cytosine (C) pyrimidine

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Overview

• Characteristics of DNA
• Chemical structure and joining of nucleotide
  subunits causes strands to differ at the ends
• One strand has a phosphate attached at the number
  5 carbon of the sugar.
• Termed the five prime (5) end
• The other strand has a hydroxyl group attached to
  the number 3 carbon of the sugar.
• Termed the three prime (3) end

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Overview

• Characteristics of DNA
• DNA occurs as double-stranded molecule
• Strands are complementary to each other
• Due to the specific base pairing of bases
• AT
• CG
• Strands are held together with hydrogen bonds
• Specific hydrogen bonding between bases
• A is bound to T by two hydrogen bonds
• G is bound to C by three hydrogen bond

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Overview

• Characteristics of DNA
• DNA molecule is antiparallel
• Strands are oriented in opposite directions
• Strands differ at the ends
• One strand oriented in the 5 to 3 direction.
• The other strand is oriented in the 3 to 5
  direction.

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Overview

• Characteristics of RNA
• RNA is made up of nucleotides
• Ribonucleotides
• RNA contains nitrogenous bases
• Adenine
• Guanine
• Cytosine
• Uracil
• Uracil replaces thymine in RNA
• RNA usually exists as single stranded molecule

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Overview

• Characteristics of RNA
• Portion of DNA acts of template for RNA synthesis
• RNA molecule called transcript
• Numerous transcripts can be produced from one
  chromosome
• Either strand of DNA can act as template
• Three different functional groups of RNA
• Messenger RNA (mRNA)
• Ribosomal RNA (rRNA)
• Transfer RNA (tRNA)

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Overview

• Regulating the expression of genes
• Nucleotide sequence codes for regulation
  mechanism for gene expression
• Mechanisms determine duration of synthesis of
  gene products
• Products are only made when required
• Key mechanism is regulation of mRNA synthesis
  from DNA
• Regulation of transcription

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DNA Replication

• DNA is replicated to create second copy of
  molecule
• Molecule is identical to original
• Replication is bidirectional
• Replication begins at specific starting point
• Proceeds in opposite directions
• Allows replication to proceed more quickly

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DNA Replication

• DNA replication
• The two strands are unwound and separated
• Free, unbound nucleotides match up to the newly
  separated nitrogenous bases of the parent strand
• The parent strand is also called the template
  strand

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DNA Replication

• DNA replication
• Base pairing is specific in DNA replication
• Where adenine is present only thymine binds in
  the new strand and vice versa
• Where guanine is present only cytosine binds in
  the new strand and vice versa
• Bases that are improperly inserted are removed
  and replaced with the correct base
• Newly added bases are added by the enzyme DNA
  polymerase

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DNA Replication

• Specifics of DNA replication
• As the strands of DNA unwind, it creates an area
  of replication called the replication fork
• As nucleotides are added, the replication fork
  moves down the parental strand

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DNA Replication

• Specifics of DNA replication
• DNA polymerase adds new nucleotides as they
  become available.
• DNA polymerase can only add nucleotides to the
  free hydroxyl at the 3 end
• DNA polymerase replicates in 5 to 3 direction
• Enzymes READS DNA template in 3 to 5 direction
• Because of the antiparallel nature of the strands
  of DNA, the two new strands will grow in opposite
  directions
• One strand is the leading strand
• One strand is the lagging strand

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DNA Replication

• Specifics of DNA replication
• Leading strand
• Is synthesized CONTINUOUSLY as the DNA polymerase
  moves towards the replication fork
• Lagging strand
• Is synthesized DISCONTINUOUSLY in pieces as DNA
  polymerase moves away from the replication fork

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DNA Replication

• Specifics of DNA replication
• DNA polymerase must bind to an RNA primer to
  begin synthesis
• A second DNA polymerase removes any RNA primers
• An RNA primer is required at each newly
  synthesized section of the lagging strand
• DNA ligase joins the fragments of the lagging
  strand

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DNA Replication

• Specifics of DNA replication
• Replication is completed when the replication
  fork reaches the end of the parent strands
• The original parent strand and the newly
  synthesized daughter strand rewind
• Each new strand of DNA consists of one parent
  strand and one daughter strand
• DNA replication is referred to as semiconservative

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Gene Expression

• Involves two separate but interrelated process
• Transcription
• Process of synthesizing RNA from DNA template
• Translation
• RNA is deciphered to synthesize protein

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Gene Expression

• Transcription
• Transcription is the synthesis of a strand of
  mRNA from a DNA template
• mRNA carries the coded information from DNA to
  the ribosome, which is the site of protein
  synthesis
• mRNA also plays an important role in translation

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Gene Expression

• Transcription
• During transcription the enzyme, RNA polymerase,
  synthesizes a complementary strand of mRNA from a
  portion of unwound DNA

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Gene Expression

• Specifics of Transcription
• RNA polymerase binds to a region of the DNA
  called the promoter
• Only one strand of DNA acts as a template
• This is called the sense strand
• The strand not transcribed is the nonsense strand

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Genet Expression

• Specifics of transcription
• Nucleotides in RNA are the same as those in DNA
  with one exception
• Thymine is replaced with uracil
• Binding in RNA is
• AU or UA
• CG or GC

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Gene Expression

• Specifics of transcription
• RNA polymerase continues down strand of DNA until
  it reaches a site on DNA called the terminator
• At the terminator RNA polymerase and the new
  strand of mRNA are released from strand of DNA

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Gene Expression

• Translation
• Translation is the decoding of information held
  in the mRNA to synthesize proteins
• Two more RNA molecules become involved in
  translation
• Ribosomal RNA (rRNA)
• Transfer RNA (tRNA)

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Gene Expression

• rRNA forms part of the ribosomal machinery used
  in protein synthesis
• rRNA builds the ribosomes
• tRNA recognizes specific sequences of mRNA and
  transports the required amino acids to form a
  polypeptide chain

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Gene Expression

• Translation
• The language of mRNA is in the form of codons
• Codons are groups of three nucleotides situated
  next to each other on DNA
• Codons are written in terms of their base
  sequence in mRNA
• The sequence of codons determines the sequence of
  amino acids in the protein

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Gene Expression

• Translation
• There are 64 codons that make up the alphabet
  of proteins
• Of the 64 codons, 61 are sense codons
• Each coding a specific amino acid
• The remaining 3 are nonsense codons
• These code for termination of the message
• Codons contained in mRNA are read into proteins
  through translation
• The site of translation is the ribosome

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Gene Expression

• In response to each codon, tRNA brings the
  appropriate amino acid to the site of translation
• Each codon has an anticodon
• The anticodon is complementary sequence to the
  codon

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Gene Expression

• Translation
• Ribosomes
• The 30s and the 50s ribosomal subunits join
  together around the mRNA
• The ribosomes direct the binding of tRNA to the
  correct codon on the mRNA
• tRNA binds to the P site and the A site of the
  50s ribosomal subunit
• The ribosomes bind to the mRNA to be translated

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Gene Expression
P site A site

• Specifics of Translation
• The first tRNA binds to a start codon in the P
  site of the ribosome
• AUG is the start codon for EVERY protein
• AUG codes for the amino acid methionine
• When the second tRNA binds to the A site, the
  amino acid of the first tRNA forms a peptide bond
  with the amino acid of the second tRNA

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Gene Expression

• Specifics of translation
• After the peptide bond is formed between the two
  amino acids, the tRNA P site leaves the ribosome
• The ribosome moves distance of one codon
• Amino acid in the A site moves to the P site
• A new tRNA fills the now empty A site

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Gene Expression

• Specifics of translation
• The ribosome continues down the strand of mRNA
• Amino acids form peptide bonds along the way
• Translation is terminated when the ribosomes come
  to a stop or nonsense codon
• At this point the ribosomes separate
• The new polypeptide chain is released
• The ribosome and the mRNA are free to begin
  translation again

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Gene Expression

• Specifics of translation
• As the ribosome moves down the strand of mRNA,
  the start codon is exposed
• Once exposed, a new ribosome will attach and
  begin another polypeptide chain

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Regulation of Gene Expression

• Microorganisms posses mechanism to synthesize
  maximum amount of cell material from limited
  energy
• Controls directed at metabolic pathways
• Two general mechanism
• Allosteric inhibition of enzymes
• Controlling synthesis of enzymes
• Directed at making only what is required

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Regulation of Gene Expression

• Principles of regulation
• Not all genes subjected to regulation
• Enzymes can be classified according to
  characteristics of regulation
• Constitutive enzymes
• Constantly synthesized
• Enzymes of glycolysis
• Inducible enzymes
• Not regularly produced
• turned on in certain conditions
• ?-galactosidase
• Repressible enzymes
• Routinely synthesized
• Generally involved in biosynthesis

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Regulation of Gene Expression

• Mechanisms controlling transcription
• Often controlled by regulatory region near
  promoter
• Protein binds to region and acts as on/off
  switch
• Binding protein can act as repressor or activator
• Repressor blocks transcription
• Activator facilitates transcription
• Set of genes controlled by protein is called an
  operon

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Regulation of Gene Expression

• Repressors
• Control mechanism that inhibits gene expression
  and decreases the synthesis of enzymes
• Repression is usually in response to the
  overabundance of an end product
• Repression decreases the rate synthesis of
  enzymes leading to the formation of the
  particular end product
• Regulatory proteins called repressors mediate
  repression
• Repressors block the ability of RNA polymerase to
  bind and initiate protein synthesis

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Regulation of Gene Expression

• Activators
• Control mechanism that turns on the transcription
  of a gene or set of genes
• Inducers are substances that act to induce
  transcription
• Enzymes synthesized in the presence of inducers
  are called inducible enzymes

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Regulation of Gene Expression

• Operon model of gene expression
• An operon is a set of genes that includes an
  operator, promoter and structural genes
• An operon is divided into two regions, the
  control region and the structural region
• The control region include the operator and the
  promoter
• This region controls transcription
• The operator acts as the on-off switch
• The structural region includes the structural
  genes
• This region contains the genes being transcribed

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Operon structure
Operator binding site for the repressor protein
for the regulation of gene expression
Promoter Binding site for RNA polymerase
Structural Genes DNA sequence for specific
proteins
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Regulation of Gene Expression

• Lac operon
• Example of induction of gene expression
• Near the operon on the DNA is a regulatory gene
  called the I gene
• This codes for the repressor protein
• When lactose is absent, the repressor protein
  binds to the operator gene
• Binding of the repressor gene prevents RNA
  polymerase from transcribing the structural genes
• No mRNA is made and no enzymes are synthesized

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Regulation of Gene Expression

• Lac operon
• When lactose is present the repressor binds to
  lactose instead of the operator
• With the repressor bound to lactose, RNA
  polymerase is able to bind to the promoter and
  transcribes the structural genes
• Lactose acts as an inducer by keeping the
  repressor from binding to the operator
• It induces the transcription of the structural
  genes

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Lac Operon
1.
2.
3.
Lactose
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Gene Expression and Environmental Fluctuations

• Many organisms adapt to changing environments by
  altering level of gene expression
• Mechanisms include
• Signal transduction
• Natural selection

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Gene Expression and Environmental Fluctuations

• Signal transduction
• Process that transmits information from external
  environment to inside cell
• Allows cell to respond to changes
• Two-component regulatory systems
• Relies on sensor and response regulator proteins
• Sensors recognize change in environment
• Response regulators activate or repress gene
  expression
• Quorum sensing
• Organisms sense density of population
• Enables activation of genes beneficial to the mass

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Gene Expression and Environmental Fluctuations

• Natural selection
• Mechanisms to enhance survivability
• Antigenic variation
• Alteration in characteristics of certain surface
  proteins
• Example Neisseria gonorrhoeae hides from host
  immunity by changing numerous surface proteins
• Phase variation
• Routine switching on and off of certain genes
• Altering expression allows portions of population
  to survive and multiply