Gene Expression Making Proteins

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Gene Expression Making Proteins

• The information content of DNA is in the form of
  specific sequences of nucleotides along the DNA
  strands (genes).
• The DNA inherited by an organism leads to
  specific traits by dictating the synthesis of
  proteins.
• Proteins are the links between genotype and
  phenotype.
• For example, Mendels dwarf pea plants lack a
  functioning copy of the gene that specifies the
  synthesis of a key protein, gibberellins.
• Gibberellins stimulate the normal elongation of
  stems.

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One gene - One Polypeptide Hypothesis

• Each gene holds the code for the production of
  one complete sequence of amino acids.
• The one polypeptide may be a functioning protein
  or enzyme by itself
• Or, it may be one subunit of a functioning protein

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Linking Gene to Protein
(or how does the information in DNA become a
gene product like blue eyes)

• Gene - A segment of DNA that specifies the amino
  acid sequence of a polypeptide
• DNA does not directly perform protein synthesis,
  instead its information is transcribed into RNA
• The specific sequence of hundreds or thousands of
  nucleotides in each gene carries the information
  for the primary structure of a protein (the
  linear order of the 20 possible amino acids).

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(No Transcript)
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RNA

• Three Classes of RNA
• Messenger RNA (mRNA)
• Takes a message from DNA to the ribosomes
• Ribosomal RNA (rRNA)
• Makes up ribosomes (along with specific proteins)
  manufacturing site of polypeptides
• Transfer RNA (tRNA)
• Transfers amino acids to ribosome to manufacture
  polypeptides

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Steps of Gene Expression

• Gene Expression (to get from DNA, written in one
  chemical language, to protein, written in
  another) requires two steps
• Transcription - a DNA strand provides a template
  for the synthesis of a complementary mRNA strand.
• Translation - the information contained in the
  order of nucleotides in mRNA is used to determine
  the amino acid sequence of a polypeptide.

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• In eukaryotic cells,
  transcription occurs
  in
  the nucleus and
  translation occurs
  mainly in the cytoplasm.
• The finished mRNA is exported to the cytoplasm.
  
• The molecular chain of
  command in a cell is
  DNA ? RNA ?
  protein.

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Transcription

• During transcription, a segment of the DNA serves
  as a template for the production of an RNA
  molecule
• Messenger RNA (mRNA)
• RNA polymerase binds to a promoter section of DNA
• DNA helix is opened so complementary base pairing
  can occur
• RNA polymerase joins new RNA nucleotides in a
  sequence complementary to that on the DNA

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• Transcription - one DNA strand (template strand)
  provides the information for ordering the
  sequence of nucleotides in a complementary RNA
  transcript.
• The RNA molecule is
  synthesized according
  to
  base-pairing rules
  (except uracil is
  substituted
  for thymine.

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Transcription of DNA to form mRNA
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• Like DNA polymerases, RNA polymerases add
  nucleotides only to the 3 end of the growing
  polymer.
• Genes are read 3-gt5, creating a 5-gt3 RNA
  molecule.
• Specific sequences of nucleotides along the DNA
  mark where gene transcription begins and ends.
• RNA polymerase attaches and initiates
  transcription at the promotor, upstream of the
  information contained in the gene, the
  transcription unit.
• The terminator signals the end of transcription.

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Stages of Transcription

• Transcription can be separated into threestages
  initiation, elongation, and termination.
• The presence of a promotor sequence determines
  which strand of the DNA helix is the template.
• Within the promotor is the starting point for the
  transcription of a gene.
• The promotor also includes a binding site for RNA
  polymerase several dozen nucleotides upstream of
  the start point.

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Initiation

• Proteins called transcription factors recognize
  the promotor region, and bind to the promotor.
• RNA polymerasebinds to transcriptionfactors to
  create atranscriptioninitiation complex.
• RNA polymerasestarts transcription.

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Elongation

• RNA polymerase moves along the DNA and untwists
  the double helix 10 to 20 bases at time.
• The enzyme addsRNA nucleotides to the3 end of
  the growing strand - elongation.
• Behind the pointof RNA synthesis,the double
  helixre-forms and the RNA molecule peels away.

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Termination

• A single gene can be transcribed simultaneously
  by several RNA polymerases at a time.
• A growing strand of RNA trails off from each
  polymerase.
• Many polymerase molecules simultaneously
  transcribing a single gene increases the amount
  of mRNA transcribed from it.
• Helps the cell make a lot of the encoded protein.
• Transcription proceeds until it reaches a
  terminator sequence in the DNA - termination.
• The pre-mRNA is cut from the enzyme.

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mRNA Processing

• Newly transcribed mRNA (pre-mRNA) in eukaryotes
  is modified while still in the nucleus before it
  is sent to the cytoplasm
• Includes leader and trailer sequences
• Includes long non-coding regions
• Modifications
• Help protect mRNA from hydrolytic enzymes
• Function as an attach here signal for ribosomes.

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Introns and Exons

• Most eukaryotic genes and their RNA transcripts
  have long non-coding stretches (introns) that lie
  between coding regions (exons). of nucleotides.
• The final mRNA transcript includes coding
  regions, exons, that are translated into amino
  acid sequences, plus the leader and trailer
  sequences.
• RNA splicing removes the introns and splices the
  entrons together to form the finished coding
  segment of mRNA.

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mRNA Processing
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(pre-mRNA)
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• RNA splicing appears to have several functions.
• First, at least some introns contain sequences
  that control gene activity in some way.
• Splicing itself may regulate the passage of mRNA
  from the nucleus to the cytoplasm.
• One clear benefit of split genes is to enable one
  gene to encode for more than one polypeptide.
• Alternative RNA splicing gives rise to two or
  more different polypeptides, depending on which
  segments are treated as exons.
• Early results of the Human Genome Project
  indicate that this phenomenon may be common in
  humans.

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• Split genes may also facilitate the evolution of
  new proteins.
• Proteins often have amodular architecturewith
  discrete structuraland functional regionscalled
  domains.
• In many cases, different exons code for
  different domains of a protein.

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Translation

• The Genetic Code
• Triplet code- each 3-nucleotide unit of a mRNA
  molecule is called a codon
• There are 64 different mRNA codons
• 61 code for particular amino acids
• Redundant code-some amino acids have numerous
  code words
• Provides some protection against mutations
• 3 are stop codons signal polypeptide termination

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Messenger RNA Codons
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Codons

• During translation, the codons are read in the
  5-gt3 direction along the mRNA.
• Each codon specifies which one of the 20 amino
  acids will be incorporated at the corresponding
  position along a polypeptide.
• Because codons are base triplets, the number of
  nucleotides in an RNA message is 3 times the
  number of amino acids in the protein product.
• It would take at least 300 nucleotides to code
  for a polypeptide that is 100 amino acids long.

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• In summary, genetic information is encoded as a
  sequence of non-overlapping base triplets, or
  codons, each of which is translated into a
  specific amino acid during protein synthesis.
• Transcription begin at the correct starting point
  on the message.
• Establishes the reading frame - subsequent codons
  are read in groups of three nucleotides.
• The cells protein-synthesizing machinery reads
  the message as a series of non-overlapping
  three-letter words.

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Transfer RNA

• tRNA transports amino acids to the ribosomes
• Single stranded nucleic acid that correlates a
  specific nucleotide sequence with a specific
  amino acid
• Amino acid binds to one end, the opposite end has
  an anticodon (complement to codon)
• the order of mRNA codons determines the order in
  which tRNAs bring in amino acids

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Transfer RNA Amino Acid Carrier
attachment site
attachment site
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• During translation, each type of tRNA links a
  mRNA codon with the appropriate amino acid.
• Each tRNA arriving at the ribosome carries a
  specific amino acid at one end and has a specific
  nucleotide triplet, an anticodon, at the other.
• The anticodon base-pairs with a complementary
  codon on mRNA.
• tRNAs deposit amino acids in the prescribed order
  and the ribosome joins them into a polypeptide
  chain

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Ribosome and Ribosomal RNA

• Ribosomes are the site of protein manufacturing.
• Each ribosome has a large and a small subunit.
• These are composed of proteins and ribosomal RNA
  (rRNA), the most abundant RNA in the cell.
• Have a binding site for mRNA and for 3 tRNAs
• Facilitate complementary base pairing
• Ribosome moves along mRNA and new tRNAs come in
  and line up in order
• Brings amino acids in line in a specific order to
  form a polypeptide
• Several ribosomes may move along the same mRNA
• Multiple copies of a polypeptide can be made
• The entire complex is called a polyribosome

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Polyribosome Structure and Function
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Stages of Translation

• Translation can be divided into three stages
  initiation elongation - termination
• All three phase require protein factors that
  aid in the translation process.
• Both initiation and chain elongation require
  energy.

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Initiation and Elongation

• Initiation brings together mRNA, a tRNA with the
  first amino acid, and the two ribosomal subunits.
• Elongation consists of a series of
  three-stepcycles as each amino acid is added on.
• During peptide bond formation, an rRNA molecule
  catalyzes the formation of a peptide bond between
  the polypeptide and the new amino acid.
• This step separates the tRNA from the growing
  polypeptide chain.

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Initiation
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Elongation
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Termination

• Termination occurs when one of the three stop
  codons reaches the amino acid binding site.
• A release factor binds to the stop codon and
  breaks the bond between the polypeptide and its
  tRNA.
• This frees the polypeptide and the translation
  complex disassembles.

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Termination
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Overview of Gene Expression
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Summary of Gene Expression
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Genes and Gene Mutations

• A gene mutation is a change in the sequence of
  bases within a gene.
• Gene mutations can lead to malfunctioning
  proteins in cells.

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Causes of Mutations

• Errors in replication
• Rare
• DNA polymerase proofreads new strands and
  errors are cleaved out
• Mutagens
• Environmental influences
• Radiation, UV light, chemicals
• Rate is low because DNA repair enzymes monitor
  and repair DNA
• Transposons
• jumping genes
• Can move to new locations and disrupt sequences

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Transposon
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Types of Mutations

• Frameshift Mutations
• One or more nucleotides are inserted or deleted
• Results in a polypeptide that codes for the wrong
  sequence of amino acids
• Point Mutations
• The substitution of one nucleotide for another
• Silent mutations
• Nonsense mutations
• Missense mutations

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DNA Technology

• Gene Cloning
• Cloning Production of many identical copies of
  an organism through some asexual means.
• Gene Cloning The production of many identical
  copies of a single gene
• Two Ways to Clone a Gene
• Recombinant DNA
• Polymerase Chain Reaction

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Cloning a Gene / Recombinant DNA
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Using Recombinant DNA Technology

• Restriction enzyme breaks open a plasmid vector
  at specific sequence of bases sticky ends
• Foreign DNA that is to be inserted is also
  cleaved with same restriction enzyme so ends
  match
• Foreign DNA is inserted into plasmid DNA and
  sticky ends pair up
• DNA ligase seals them together

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Restriction Enzymes and Sticky Ends
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Polymerase Chain Reaction

• Amplifies a targeted DNA sequence
• Requires DNA polymerase, a set of primers, and a
  supply of nucleotides
• Primers are single stranded DNA sequences that
  start replication process
• Amount of DNA doubles with each replication cycle
• Process is now automated (repeated cycling caused
  logrhythmic increases in concentration of copied
  DNA sequence

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Polymerase Chain Reaction (PCR)
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DNA Fingerprinting

• Permits identification of individuals and their
  relatives
• Based on differences between sequences in
  nucleotides between individuals
• Detection of the number of repeating segments
  (called repeats) are present at specific
  locations in DNA
• Different numbers in different people
• PCR amplifies only particular portions of the DNA
• Procedure is performed at several locations to
  identify repeats

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DNA Fingerprints
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Biotechnology

• Biotechnology uses natural biological systems to
  create a product or to achieve a goal desired by
  humans.
• Transgenic organisms have a foreign gene inserted
  into their DNA

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Transgenic Bacteria

• Medical Uses Production of Insulin, Human
  Growth Hormone, Tissue Plasminogen Activator,
  Hepatitis B Vaccine
• Agricultural Uses Bacteria that protects plants
  from freezing, bacteria that protect plant roots
  from insects
• Environmental Bacteria that degrade oil (clean
  up after oil spills), bacteria that remove sulfur
  from coal

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Transgenic Plants

• Plants have been engineered to secrete a toxin
  that kills insects
• Plants have been engineered to be resistant to
  herbicides

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Transgenic Animals

• Fish, cows, pigs, rabbits and sheep have been
  engineered to produce human growth hormone in
  order to increase size of the animals

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Transgenic Animals