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Chapter 16
DNA and Its Role in Heredity
The Search for the Genetic Material
• Chromosome made of
– Proteins
– DNA
• Frederick Griffith (1928)
– He studied Streptococcus pneumoniae
• A bacterium that causes pneumonia in mammals.
• R strain was harmless
• S strain was pathogenic
– Smooth, enabled the pathogen to get into the cell
Living S Strain
Living R Strain
Heat Killed S Strain
Heat Killed S Strain and Living R Strain
Living S cells found in blood
sample from dead mouse
• Heat treated S strain
• Didn’t kill the DNA completely
• only transformed
• Heat opened up DNA
• R strain took in small S strain strands
• turned into live S - lethal
• Griffith called this process transformation
• Change in genotype and phenotype
• Assimilation of external DNA
• For the next 14 years scientists tried to identify
the transforming substance
• Avery, McCarty, MacLeod (1944)
– Isolated different parts of the bacteria and injected
each part into a mouse
• Carbohydrates
– Mouse lives
• Lipids
– Mouse lives
• Proteins
– Mouse lives
• Nucleic Acids
– Mouse dies
• Still many biologists were skeptical
• Hershey, Chase (1952)
– Transduction
• Infiltrate into cell
• Replicate until the cell bursts
– Lytic cycle
– Worked with viruses that infect bacteria
• Bacteriophages or phages
– Virus
• DNA (or RNA) center
– Program host
– Restriction enzyme
» Cuts up the host cell DNA
• Protein coat
– Labeled the protein or DNA with radioactive isotopes
• Sulfur protein
• Phosphorous DNA
– Tracked which entered the E. coli cell during infection
Pellet = trans infected bacteria
Only DNA could get into the bacteria
Protein left outside
• What they knew about the structure of DNA
– DNA was a polymer of nucleotides
• Phosphate group
• Deoxyribose
• Nitrogenous base
– Adenine, guanine, thymine, cytosine
• Chargaff (1947)
– DNA composition changes between different
organisms
– Chargraff’s rule
• Molecular diversity
• %T = %A
• %G = %C
• In every organism he studied
• Wilkins, Franklin (1950’s)
– X-ray crystallography
• DNA helical structure
• Estimate the width of the helix
– Could only be double stranded
• Linus Pauling
• Watson, Crick (1950’s)
– Model of Double Helix DNA
• Full turn every 3.4 nm
• 10 layers of base pairs per turn
• Nitrogenous bases hydrophobic
– Shielded by sugar back bone from aqueous medium
– 2nm helix diameter indicated by the X-ray data
• Pyrimidine-purine pairing
• Adenine and Guanine = purine
• Cytosine and Thymine = pyrimidine
• The base-pairing rules
– A - T 2 hydrogen bonds
– G - C 3 hydrogen bonds
• This does not restrict the sequence of nucleotides
– The linear sequence varied in countless ways
• Each gene has a unique order of nitrogen bases
• April 1953 - Watson and Crick published a succinct
paper in Nature on the double helix model of DNA
DNA Replication
• Base pairing enables existing DNA strands to
serve as templates for new complementary
strands
– Each strand can form a template when separated
• Semiconservative replication
– Each of the daughter molecules will have one old
strand and one newly made strand
Conservative
Semi-Conservative
Dispersive
• Meselson, Stahl (1950’s)
– They labeled the nucleotides of the old strands with a
heavy isotope of nitrogen (15N), while any new
nucleotides were indicated by a lighter isotope (14N)
– Replicated strands could be separated by density in a
centrifuge
• New strands less dense
• Origins of replication
– Special sites where DNA replication begins
– Bacteria
• Single site
– Both directions
– Eukaryote
• Hundreds or thousands per chromosome
• DNA polymerase
– Enzyme that adds new nucleotides to the
growing strand
• Driven by nucleoside triphosphates
– Similar to ATP
» Suga component is deoxyribose instead of ribose
» Loses a pyrophosphate - 2 phosphate groups
» Hydrolysis supplies energy for polymerization
• antiparallel
• The strands of the helix are antiparallel
– DNA polymerases can only add nucleotides to the
free 3’ end of a growing DNA strand
– A new DNA strand can only elongate in the 5’→3’
direction
– Leading strand
• 3’→5’ parental strand
• Can be used as a template for a continuous complementary
strand
– Lagging Strand
• 5’→3’ parental strand
• Copied away from the fork in short segments
– Okasaki fragments
• DNA ligase
– Joins together fragments
– sealing repairs
– sealing recombination fragments
• Priming
– DNA polymerase cannot initiate synthesis
• Can only add nucleotides to an existing chain
– Primase
• RNA polymerase
• Adds a short segment of RNA
– DNA polymerase can now add nucleotides to the
primer
– The primer is later replaced with DNA nucleotides
– Leading strand
• Requires only one primer
– Lagging strand
• Requires each fragment be primed
• Helicase
– Untwists and separates DNA strands
• Single strand binding proteins
– Helps separate and prevent ssDNA from reforming
• Topoisomerase
• Single strand breaks to allow for DNA
unwinding
• Prevent supercoiling
• Strand breakage during
recombination
• Replication factories
• Mechanisms create a machine
• Stationary – fixed by the nuclear matrix
• Spits out daughter strands
Enzymes Proofread and Repair
• DNA polymerase proofreads each new nucleotide
against the template as soon as it is added
– If there is an incorrect pairing, the enzyme removes the wrong
nucleotide and then resumes synthesis
• Mutations that occur after DNA synthesis is completed
can often be repaired
• Nucleotide excision repair
– Nuclease cuts out a segment of a damaged strand
– The gap is filled in by DNA polymerase and ligase
• Exonuclease
– Enzyme that removes nucleotides from the end of a
polynucleotide
• Has direction
• Used to edit DNA, remove
RNA primers
• Related to Endonuclease
Chromosome Ends
• Telomeres
– Ends of eukaryotic chromosomal DNA molecules
– Special nucleotide sequences
• TTAGGG
• Repeated between 100 and 1,000 times
– Protect genes from being eroded
• Telomerase
– Uses a short molecule of RNA as a template to
extend the 3’ end of the telomere
– Present in germ-line cells
Chapter 17
From DNA to Protein:
Gene Expression
DNA
• ATCG
• Nucleic acid
• Double helix
• Generally stays in nucleus
– Storing and transferring
genetic information
• Stable under alkaline
conditions
• 5 carbon sugars
– Deoxyribose
• Only an -H on its second
carbon
RNA
• AUCG
• Nucleic acid
• Single strand
• Outside the nucleus
– Codes for amino acids
– Messenger btw DNA/ribosomes
• Unstable under alkaline
conditions
• 5 carbon sugars
– Ribose
• -OH group attached to second
carbon
Gene to protein
• Archibald Garrod
– Genes dictate phenotypes thru enzymes
• Diseases reflect inability to produce an enzyme
• Beadle and Tatum
– Mutant bread mold in mimimal medium + 1
animo acid
– One gene – one enzyme
• Amended to one gene – one polypeptide
An Overview
• Genes provide the instructions for making
specific proteins
• The bridge between DNA and protein synthesis
is RNA
• To get from DNA to protein there are two major
stages
– Transcription
• DNA provides a template to make RNA
– Translation
• Information contained in RNA nucleotides determines amino
acid sequence
Transcription and translation
Pro
• Very quick transcription to
translation
– Basically simultaneously
• All occur in Cytoplasm
• mRNA directly
transcribed from DNA
Eu
• Much slower translation
and transcription
– Different space and time
• Transcription in nucleus
• Translation in cytoplasm
• Pre - mRNA
– RNA processing into
mature mRNA
http://pediaa.com/difference-between-prokaryotic-and-eukaryotic-transcription/
Transcription
• Messenger RNA is transcribed from the template
strand of a gene
• Prolene
– CGG
– Found in every living thing
• In DNA or RNA, the
four nucleotides act
like the letters of the
alphabet
• Triplet code
– Three consecutive
bases specify an
amino acid
• Unit called codon
– 61 of 64 triplets code
for amino acids
– AUG
• Methionine
• Also, start of translation
– 3 stop codes
• UAA, UAG, UGA
• RNA polymerase
– Separates the DNA strands
– Bonds the RNA nucleotides
– Can add nucleotides only to the 3’ end of the growing
polymer
• Genes are read 3’→5’
• Creating a 5’→3’ RNA molecule
• Specific sequences of nucleotides along the DNA
mark where gene transcription begins and ends
– The transcription unit
• Part of DNA transcribed into RNA
• The promotor
– Where the RNA polymerase attaches
» Will not work if attached inproperly
– “Upstream” of the transcription unit
• The terminator
– Signals the end of transcription
• Transcription can be separated into three stages:
– Initiation
– Elongation
– Termination
Initiation
• Promoter
– Transcription start point
• Nucleotide where RNA synthesis begins
– TATA box
• DNA sequence upstream of start point
– Transcription factors
•
•
•
•
Collection of proteins
Recognizes TATA
Only certain ones RNA poly binds to
Transcription initiation complex = TF and RNA poly
Elongation
• RNA poly
– Untwists
• Exposes 10 – 20 bases
– Adds nucleotides to the 3’ end of the growing strand
• Behind the point of RNA synthesis
– DNA rewinds
– RNA peels away
– 60 nucleotides per sec
• A single gene can be transcribed multiple times
– Mass production of proteins
Termination
• Pro
– Stops at the termination signal
– Releases both DNA and RNA
• Eu
– Continues ~ 100 nucleotides beyond termination
signal
• AAUAAA in pre-mRNA
• Stop codon only finishes translation
– Termination signal after stop codon finishes transcription
– Pre-mRNA cut free 10-35 nu past TS
RNA Processing
• Prokaryotic cells
– Many proteins synthesized at a time
• Similar to each other
– RNA after transcription is mRNA
• Eukaryotic cells
– Gene only codes for 1 protein
– RNA formed after transcription is pre-mRNA
• Needs to be modified
– Ends modified to prevent degradation from hydrolytic
enzymes
– 5’ cap
• Modified guanine (guanine triphosphate)
• Guides attachment of the polypeptide
– Poly-A tail
• 50 – 250 adenine
• Assists in export from nucleus
• Nucleoplasm to cytoplasm
– RNA splicing
• Covalently altered
• Long noncoding stretches of nucleotides
– Introns
» Noncoding segments between coding segments
– Exons
» Exit nucleus
» Includes leader/trailer segments and translated segments
• RNA splicing removes introns and joins exons to create an
mRNA molecule with a continuous coding sequence
Leader
segment help
recognize
start codon
Trailer
segment
• Pre-mRNA HnRNA
– Before introns are cut out
• Spliceosome
– SnRNPs
• Recognize nucleotide segments on the ends of introns
• Cuts points on the RNA, releases the intron and joins the
flanking portions
• Catalytic process
• Ribozymes
– Self splicing in ribosomal RNA
– Catalysts
• Alternative RNA splicing/more possibilities for
crossing over
Translation
• Transfer RNA (tRNA)
– Transfers amino acids from
the cytoplasm to the
ribosome
– Anticodon to polypeptide
• Hydrogen bonds to the mRNA
– 45 types
• Anticodon can recognize
more than 1 codon
• Wobble
– Relaxed 3rd base
– U can bond with A or G
– Inosine, anticodon, can bond
with U, C, or A
• Aminoacyl-tRNA
synthetase
– Enzyme
• Join amino acid to correct
tRNA
– 20 different types
• One for each amino acid
• Specific combination at
active site
– Powered by hydrolysis of
ATP
– Creates activated animo
acid
Fig. 17.14
• Ribosomes
– Large and small subunit
• Formed in the nucleolus
– Composed of proteins and 60% rRNA
– A binding site for mRNA
– Three binding sites for tRNA molecules.
• The P site
– Holds the tRNA carrying the growing polypeptide chain
• The A site
– Carries the tRNA with the next amino acid
– Holds amino acid to carboxyl
– Catalyzes peptide bond
• The E site
– tRNAs leave the ribosome
• Translation can be divided into three
stages:
– Initiation
– Elongation
– Termination
• All three phase require protein “factors”
• Initiation and elongation require energy
– GTP
• Initiation
– mRNA
– A tRNA with the first amino acid
– The two ribosomal subunits
• Initiation factors
– MET
• Always first
Fig. 17.17
• Elongation
– Series of 3 steps
• Codon recognition
• Peptide bond formation
• Translocation
Peptidyl
transferase
• Termination
– Stop codon reaches the A site
– Release factor
• Binds to the stop codon
• Adds a water molecule
• Hydrolyzes the bond between the polypeptide and
its tRNA in the P site
Fig. 17.19
• Polyribosomes
– Strings of ribosomes
– Make copies of a polypeptide quickly
Signal Peptides
Comparing Prokaryote and Eukaryote
• Prokaryotes can transcribe and translate
the same gene simultaneously
• RNA processing
• RNA polymerases differ and eukaryotes
require transcription factors.
• Their ribosomes are also different
Mutations
• Changes in the
genetic material
• Point mutation
– Base pair substitution
• Missense mutations
• silent mutations
• Nonsense mutations
– Non-functional protein
Fig. 17.24
Mutations
• Changes in the
genetic material
• Point mutation
– Base pair substitution
• Silent mutations
• Missense mutations
• Nonsense mutations
– Insertions / deletions
• Frameshift mutations
Fig. 17.24
Other things to know
•
•
•
•
RNA modification
Types of bonding in DNA
Structure of ribosomes
Types of RNA