Unit 6: From Gene to Protein

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Unit 6 From Gene to Protein

• Chapters 17 20
• Campbell Biology, AP Edition
• Beth Walker

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Metabolism Genes

• Non-functional enzymes (proteins) can lead to
  metabolic defects
• A study of metabolic diseases suggested that
  genes specify proteins
• Alkaptonuria ? black urine from alkapton
• PKU (phenyketonuria)
• Genes dictate phenotype

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One Gene-One Enzyme Hypothesis

• Beadle Tatum (1941)
• Compared different nutritional mutants of bread
  mold (Neurospora)
• Created mutations by X-ray treatments
• Wildtype grows on minimal media
• Each mutant requires different
• amino acids
• Each type of mutant lacks a certain
• enzyme needed to produce a
• certain amino acid
• Broken gene ? results in non-functional enzyme

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Beadle Tatums Experiment
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One Gene One Polypeptide

• One Gene One Enzyme was modified
• not all proteins are enzymes
• Those proteins are coded for by genes too
• One Gene One Protein
• But many proteins are composed of several
  polypeptide chains (quaternary structure) each
  polypeptide chain has its own gene
• One Gene- One Polypeptide

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Central Dogma

• Transcription and
• Translation are the
• 2 processes linking
• a gene to a protein

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Theres a problem.

• Where are the genes?
• On chromosomes in the nucleus of the cell
• Where are proteins made?
• On the ribosome, in the cytoplasm of the cell
• How does the information that codes for genes get
  from the nucleus to the cytoplasm?
• Messenger RNA (mRNA)

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RNA vs. DNA

• Ribose sugar (C5H10O5)
• Nitrogen Base uracil, instead of thymine
• U A
• C G
• Single stranded
• transcription
• DNA RNA

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Transcription

• One side of the DNA
• is the template
• Which one?
• The mRNA is complementary
• to the DNA
• RNA polymerase is the
• enzyme involved

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Transcription Initiation

• TRANSCRIPTION INITIATION
• COMPLEX made up of .....
• 1) TATA Box in located in the
• promoter sequence, upstream
• from the start point
• 2) Transcription Factor
• proteins that assist
• in initiation recognize the
• TATA box
• 3) Additional transcription factors join
• 4) RNA polymerase binds
• to a promoter sequence
• on DNA

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Transcription Elongation

• Role of the Promoter
• Initiation site ( TATA box)
• Which strand to read
• Direction on DNA
• Reads DNA 3 ? 5
• RNA polymerase II
• unwinds DNA
• Reads DNA 3 ? 5
• Builds RNA 5 ? 3
• No proofreading

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Transcription Elongation

• One gene can be
• transcribed by
• multiple RNA
• polymerases ?
• help the cell make
• more of the protein

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Transcription Termination

• RNA polymerase stops
• at the termination sequence
• Prokaryotes stops at the
• end of the stop signal
• Eukaryotes stops
• hundreds of nucleotides
• past the stop signal
• at an AAUAAA sequence
• mRNA leaves the nucleus
• through small pores in the
• nucleus (called pre mRNA)

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Transcription Differences Prokaryotes vs.
Eukaryotes

• Time (longer in eukaryotes)
• Physical separation between processes in
  eukaryotes
• RNA processing in eukaryotes
• Prokaryotes have one RNA polymerase while
  eukaryotes have RNA polymerase I, II, III
• RNA polymerase II used to make mRNA

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RNA Processing

• Add a 5 guanine cap ? protects mRNA from being
  degraded by enzymes serves as an attachment
  recognition site for the ribosome
• 3 Poly A Tail ? also protects mRNA, assists in
  attachment to ribosome, helps remove mRNA from
  the nucleus

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RNA Splicing

• Introns ? non-coding segments of mRNA
• Exons ? coding segments of mRNA will be
  expressed into a protein
• Introns are spliced out of the RNA by the
  spliceosome
• Spliceosome ? made up of.
• Small nuclear ribonucleoproteins (snRNPs)
  (recognize splice sites)
• Other proteins

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RNA Splicing
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Transcription Factors

• Proteins which bind
• to DNA turn on or
• off transcription
• master regulators
• Genes controlling
• development
• -We will learn more about gene regulation in the
  next unit

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The Genetic Code

• Nirenburg Matthaei
• Determined 1st codon amino acid match
• UUU coded for phenylalanine
• Created artificial poly U mRNA
• Added mRNA to test tube of ribosomes
  nucleotides
• mRNA made a single amino acid polypeptide chain
• phe-phe-phe-phe-phe-phe

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The Genetic Code

• Code is the same for all organisms
• Code is redundant ? several codons code for each
  amino acids
• Start Codon
• AUG ? methionine
• Stop Codon
• UGA, UAG, UAA

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The Genetic Code

• How are the codons read?
• Triplet Code
• DNA 3 TAC GCA CAT TTA CGT ACG CGG5
• mRNA 5 AUG CGU GUA AAU GCA UGC GCC3
• tRNA 3UAC5
• Met
• (protein) GCA
• Arg
• CAU
• Val

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Translation - Overview

• Ribosome reads mRNA
• in codons
• tRNA brings in the
• correct amino acid
• Amino acid from tRNA
• (anticodon) is
• complementary to the
• mRNA (codon)
• Peptide bonds link the
• amino acids together to
• form a polypeptide chain

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tRNA Molecule

• Anticodon on the end of the clover leaf
• Amino acid attached to the 3 end
• Made in the nucleus
• Cell keeps the cytoplasm supplied with tRNA
• May be used repeatedly

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tRNA Molecule

• Anticodon 3 to 5
• Codon 5 to 3
• Wobble Effect some
• tRNA anticodons can
• recognize more than
• codon due to flexibility
• of the 3rd base
• Codons for each amino
• acid usually only differ
• in the 3rd base

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Aminoacyl tRNA Synthetase

• Enzyme that bonds an amino acid to a tRNA
• Endergonic rxn
• Energy stored in
• tRNA- amino acid bond
• 20 enzymes ? one per amino acid

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Ribosome

• Structure
• Ribosomal RNA proteins
• 2 subunits
• Large
• Small

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Ribosome

• P site holds tRNA carrying the growing
  polypeptide chain
• A site holds tRNA carrying the next amino acid
  to be added
• E site where the tRNA will leave the ribosome

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Initiation of a Polypeptide Chain

• Brings together mRNA, ribosome subunits,
  proteins, and initiator tRNA

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Elongation of Polypeptide Chain
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Elongation Continued

• Codon Recognition mRNA in A site forms H bonds
  w/ anticodon of tRNA with the correct amino acid
• Peptide Bond Formation formed b/w the amino
  acid in the P site and the one in the A site
• Translocation A site tRNA moves to the P site
  P site tRNA moves to the E site

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Termination of a Polypeptide Chain

• Stop codon reaches the A site (UGA, UAG, UAA)
• Release factor (protein) bonds to A site
• Adds a water molecule, instead of an amino acid,
  to the polypeptide chain

Now.what will happen to the polypeptide chain?
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(No Transcript)
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Polyribosomes

• Many ribosomes read a single mRNA simultaneously
  making many copies of a protein

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Prokaryotes can have simultaneous
transcription and translation!
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Protein Targeting

• Signal Peptide
• Address Label
• Sends the ribosome
• to attach to the ER
• Destinations?
• Secretion
• Nucleus
• Mitochondria
• Chloroplasts
• Cell membrane
• Cytoplasm

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Nitrogen Base Mutation-Point Mutations

• 1 base pair change
• Base-pair substitution
• Silent no AA change
• Missense change AA
• Nonsense change a stop codon

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Nitrogen Base Mutation - Frameshift Mutations

• Insertion
• Adding base(s)
• Deletion
• Removing base(s)

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Molecular Basis of Sickle- Cell Anemia
What type of mutation is this?
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Biotechnology

• Manipulation of organisms to make products
• Genetic Engineering
• Manipulation of DNA
• Need a set of special tools to engineer DNA,
  genes, organisms
• What are some of those tools?

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Genetic Engineering Tools

• Basic Tools
• Restriction Enzymes
• Ligase
• Plasmids/Cloning
• Advanced Tools
• PCR
• DNA Sequencing
• Gel Electrophoresis
• Southern Blotting

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How are the Basic Tools Used?

• A) Cut
• Using Restriction Enzymes
• B) Paste
• Using DNA Ligase
• C) Copy
• Using Plasmids/Cloning
• Using PCR
• D) Find
• Using Southern Blotting/Probes

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Cutting DNA

• Restriction Enzymes
• Evolved in bacteria to cut up foreign DNA
• Added protection against viruses other bacteria
• Hundreds of different enzymes
• Cut at restriction sites (specific sequence of
  DNA)
• Palindrome (RACECAR)
• Produces sticky ends or blunt ends

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Cutting DNA

• Restriction Enzymes Continued.
• Named for the organism that they come from
• Example EcoRI ? 1st restriction enzyme
• found in E.coli
• The cutting process leaves either sticky or
  blunt ends

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Pasting DNA

• Sticky Ends
• H bonds form b/w complementary bases
• Ligase
• Enzyme seals strands

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Copying DNA

• Plasmids
• small, self-replicating
• circular DNA molecule
• - found in bacteria
• used to insert DNA
• sequence
• acts as a vector
• Transformation
• Insert recombinant plasmid into bacteria move
  genes of interest into the bacteria using the
  plasmid as a vector

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Plasmids Cloning A Gene

• Isolate vector (plasmid) gene source
• Insert DNA of interest into the vector (plasmid)
• Introduce the cloning vector (plasmid) into the
  cell
• Clone the cells the foreign gene
• ID the cell clones carrying the gene of interest

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Gene Cloning
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How can we make copies of DNA?

• Cloning
• Polymerase Chain Reaction (PCR)
• A small piece of DNA can be quickly copied
• Only 1 cell of DNA needed to start
• Make billions of copies of a sequence of DNA
• Very fast
• High specificity
• Developed in 1985 by Kary Mullis
• Used to makes copies of
• Ancient DNA
• DNA at crime scenes
• DNA from embryos to detect genetic disorders

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Polymerase Chain Reaction
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PCR Primers

• Uses Taq polymerase
• From hot springs bacteria Why is this important?
• Primers are important
• Identify and flank the target sequence

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Gel Electrophoresis

• Separating fragments of DNA by size
• DNA has a charge will move toward the
  electrode
• agarose gel
• consistency of jello
• Jungle Analogy small fragments move further
  faster

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Gel Electrophoresis

• Size of fragment is inversely proportional to the
  distance moved!

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Gel Electrophoresis Results

• Used to determine paternity
• Used to place suspect at the
• scene of the crime

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RFLP

• Restriction Fragment Length Polymorphism
• The differences in a DNA sequence on chromosomes
  can result in different patterns of DNA fragments
• Different banding patterns are created
• Useful as a genetic marker for making linkage
  maps
• Detected analyzed by Southern blotting

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Southern Blotting

• Want to locate a sequence on a gel?

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Southern Blot

• Transfer DNA from gel to filter paper
• Capillary action pulls a basic solution up the
  gel and sheet of paper
• Paper is exposed to a solution with a
  radioactively labeled probe
• Probe attaches (by base-pairing) to restriction
  fragments
• Film is laid over the paper radioactivity in
  probe forms an image on the film

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DNA Sequencing Sanger Method

• Entire genomes of
• organisms can be mapped!
• Developed by
• Frederick Sanger in 1978
• Sanger received the
• Nobel Prize in 1980
• Mostly automated today!

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Human Genome Project

• US Govt Project
• Started in 1990
• Estimated to take 15 years
• Department of Energy
• National Institutes of Health
• Started by Jim Watson
• Led by Francis Collins
• Celera Genomics (private company)
• Craig Venter
• Challenged the govt
• Said it could complete the genome faster

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Human Genome Project
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Human Genome Project
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Mapping the Genome Different Approaches

• Map-Based
  Shot Gun
• Govt Approach
  Celera Genomics

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Human Genome Project

• June 26, 2001 Published
• a working draft of the
• DNA sequence of the
• human genome
• GenBank public accessible
• genetic sequence database
• for all DNA sequences

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GenBank Growth
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Recombinant DNA

• Combining sequences of DNA from 2 different
  sources
• Human insulin gene into bacteria
• Frost resistant gene from Arctic fish into
  strawberries
• Green glow gene in our E.coli bacteria

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Applications of DNA Technology

• Medical diagnostics
• Diseases genetic disorders before birth
• Gene therapy
• Medical treatment
• Pharmaceutical production
• Human insulin growth hormones
• Forensics
• DNA Fingerprint identify the guilty
• Environmental Cleanup
• Extract metals, clean up waste (oil spills)
• Agricultural applications
• Food with more nutrients, pest-resistant
• Ethics
• Are genetically modified organisms safe? What
  about natural selection?

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Gene Therapy