Structure and Replication of DNA...DNA Replication: Semiconservative Replication- DNA unzips and a...
Transcript of Structure and Replication of DNA...DNA Replication: Semiconservative Replication- DNA unzips and a...
Structure and Replication of DNA
John Kyrk Animations
• http://www.johnkyrk.com/DNAanatomy.html
Are Genes Composed of DNA or Protein?
• DNA
– Only four nucleotides
• thought to have monotonous structure
• Protein
– 20 different amino acids – greater potential variation
– More protein in chromosomes than DNA
Bacterial Transformation Experiments
Fredrick Griffith (1928) –demonstrate the existence of “Transforming Principle,” a substance able to transfer a heritable phenotype (trait) from one strain of bacteria to another.
Avery MacLeod and McCarty – determine the
transforming principle was DNA.
Streptococcus Pneumoniae
Griffith Experiment
Avery Experiment
Viruses Injecting DNA into a Bacterium
Bacterial cell
Phage head
Tail sheath
Tail fiber
DNA
10
0 n
m
Hershey Chase Experiment – Viruses can be used to transfer traits and therefore DNA
Traits can be transferred if DNA is transferred.
(a) Tobacco plant expressing a firefly gene
(b) Pig expressing a jellyfish gene
Additional Evidence • Chargaff Ratios
• % A = %T and %G = %C (Complexity in DNA Structure)
A T G C
Arabidopsis 29% 29% 20% 20%
Humans 31% 31% 18% 18%
Staphlococcus 13% 13% 37% 37%
• DNA Content of Diploid and Haploid cells – Haploid cells contain half of the amount of DNA
Gametes Somatic Cells
Humans 3.25pg 7.30 pg
Chicken 1.267pg 2.49pg
DNA
Friedrich Meischer (1869) extracted a phosphorous rich material from nuclei of which he named nuclein
DNA – deoxyribonucleic acid - Monomer – Nucleotide
Deoxyribose Phosphate Nitrogenous Base (4 types – 2
purines G & A; 2 pyrimidines T & C)
- Phosphodiester Bond linkage - DNA has direction - 5’ and 3’ ends - Chromosomes are composed of DNA
Fig. 16-UN1
Purines have two rings. Pyrimidines have one ring.
Purine + purine: too wide
Pyrimidine + pyrimidine: too narrow
Purine + pyrimidine: width consistent with X-ray data
Watson and Crick Model • Franklins X-Ray Data
– DNA is Double Helix • 2 nm diameter
• Phosphates on outside
• 3.4 nm periodicity
• Bases 0.34nm apart
• Watson and Crick
– Base Pairing- Purine with Pyrimidine (A/T & C/G)
DNA double helix (2 nm in diameter)
Nucleosome (10 nm in diameter)
Histones Histone tail
H1
DNA, the double helix Histones Nucleosomes, or “beads on a string” (10-nm fiber)
DNA Structure – Chromatin = unwound DNA
video
Chromatin coils around proteins to form Chromosomes
30-nm fiber
Chromatid (700 nm)
Loops Scaffold
300-nm fiber
Replicated chromosome (1,400 nm)
30-nm fiber Looped domains (300-nm fiber)
Metaphase chromosome
30 nm chromatin fiber
1. Held together by histone tails interacting with neighboring nucleosomes 2. Inhibits transcription 3. Allows DNA replication
DNA Replication:
Semiconservative Replication- DNA unzips and a new strand builds on the inside. The new strands each have a
piece of the “old” DNA
Other Models of Replication
Conservative Replication
Semi-Conservative Replication
Dispersive Replication
Culture Bacteria in 15N isotope (DNA fully 15N)
One Cell Division in 14N
2nd Cell Division in 14N
Less Dense More Dense
Density Centrifugation
15N DNA 15N/14N DNA
15N/14N DNA
14N DNA
DNA Replication: A Closer Look
• The copying of DNA is remarkable in its speed and accuracy
• More than a dozen enzymes and other proteins participate in DNA replication
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
• Replication bubbles are the “unzipped” sections where replication occurs all along the molecule
• At the end of each replication bubble is a replication fork: a Y-shaped region where new DNA strands are elongating
• Helicase: enzyme that unzips the double helix at the replication forks
• Single-strand binding protein binds to and stabilizes single-stranded DNA until it can be used as a template
• Topoisomerase corrects “overwinding” ahead of replication forks by breaking, swiveling, and rejoining DNA strands
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Origins of Replication Video
Fig. 16-13
Topoisomerase
Helicase
Primase Single-strand binding
proteins
RNA
primer
5 5
5 3
3
3
DNA Polymerase – enzyme that builds the new strand
3’ 5’
3’ 5’ Pol
Pol
Leading and Lagging Strands – Polymerase only works on the 3’ to 5’ DNA side. Must do the 5’ to 3’ side in
segments called Okazaki fragments. 3’ to 5’ = Leading (easy) strand; 5’ to 3’ = lagging (segmented) strand
5’
5’
3’
3’
Leading Strand
Lagging Strand
Pol
3’
5’
RNA Primer
Video
Other Proteins at Replication Fork
Pol
5’
5’
3’
3’
Leading Strand
Lagging Strand
Pol
3’
5’
Helicase
Single Stranded Binding Proteins
Primase
DNA Pol I
Ligase
DNA Pol III
Lagging strand
assembly and
Okazaki
fragments
Overview
Origin of replication
Leading strand
Leading strand
Lagging strand
Lagging strand
Overall directions
of replication
Template
strand
RNA primer
Okazaki
fragment
Overall direction of replication
1 2
3
2
1
1
1
1
2
2
5
1 3
3
3
3
3
3
3
3
3
5
5
5
5
5
5
5
5
5
5
5 3
3
Damaged DNA Nuclease Excision Repair – cut and replace
Nuclease
DNA Polymerase
Ligase
Replicating the Ends of DNA Molecules
• Limitations of DNA polymerase create problems for the linear DNA of eukaryotic chromosomes
• The usual replication machinery provides no way to complete the 5 ends, so repeated rounds of replication produce shorter DNA molecules
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Replicating Ends of Linear Chromosomes
Fig. 16-19
Ends of parental
DNA strands Leading strand
Lagging strand
Lagging strand
Last fragment Previous fragment
Parental strand
RNA primer
Removal of primers and
replacement with DNA
where a 3 end is available
Second round
of replication
New leading strand
New lagging strand
Further rounds
of replication
Shorter and shorter daughter molecules
5
3
3
3
3
3
5
5
5
5
• If chromosomes of germ (sex) cells became shorter in every cell cycle, essential genes would eventually be missing from the gametes they produce
• An enzyme called telomerase catalyzes the lengthening of telomeres in germ cells; it adds temporary DNA so the strand can be completed
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Telomerase
Telomeres
1 µm
END STRUCTURE/REPLICATION
• Crash Course Video
• DNA Activities
Chapter 10 From Gene to Protein
Protein Synthesis: overview
One gene-one enzyme hypothesis (Beadle and Tatum)
One gene-one polypeptide (protein) hypothesis
Transcription: synthesis of RNA under the direction of DNA (mRNA)
Translation: actual synthesis of a polypeptide under the direction of mRNA
The “Central Dogma” Flow of genetic information in a cell
How do we move information from DNA to proteins?
replication
protein RNA DNA trait
DNA gets all the glory, but proteins do all the work!
mRNA
From gene to protein
DNA transcription
nucleus cytoplasm
aa
aa
aa
aa
aa
aa
aa
aa
aa
aa
aa
protein translation
ribosome
trait
Genetic Code
Identifying Polypeptide Sequence
• Locate start codon (1st AUG from 5’ end)
• Identify Codons (non overlapping units of three codons including and following start codon)
• Stop at stop codon (remember stop codon doesn’t encode amino acid)
• Nucleotides before start codon – 5’UTR – untranslated region
• Nucleotides after stop codon -3’UTR
• [MetArgAsnAlaSerLeu]
GACGACGGAUGCGCAAUGCGUCUCUAUGAGACGUAGCUCAC 5’
The Genetic Code
•Use the code by reading from the center to the outside •Example: AUG codes for Methionine
Name the Amino Acids
• GGG?
• UCA?
• CAU?
• GCA?
• AAA?
Central Dogma of Molecular Biology
Transcription
from
DNA nucleic acid language
to
RNA nucleic acid language
RNA ribose sugar
N-bases
uracil instead of thymine
U : A
C : G
single stranded
lots of RNAs
mRNA, tRNA, rRNA, siRNA…
RNA DNA transcription
Transcription Making mRNA
transcribed DNA strand = template strand
untranscribed DNA strand = coding strand same sequence as RNA
synthesis of complementary RNA strand transcription bubble
enzyme RNA polymerase
template strand
rewinding
mRNA RNA polymerase
unwinding
coding strand
DNA C C
C
C
C
C
C
C
C C C
G
G G
G
G G
G G
G
G
G A
A
A A A
A
A
A
A
A A
A
A T
T T
T
T
T
T
T
T T
T
T
U U
5
3
5
3
3
5 build RNA 53
Animation of Transcription
• http://vcell.ndsu.nodak.edu/animations/transcription/movie-flash.htm
RNA polymerases 3 RNA polymerase enzymes
RNA polymerase 1
only transcribes rRNA genes
makes ribosomes
RNA polymerase 2
transcribes genes into mRNA
RNA polymerase 3
Makes tRNA
each has a specific promoter sequence it recognizes
Which gene is read? Promoter region
binding site before beginning of gene
TATA box binding site
binding site for RNA polymerase
& transcription
factors (helpers)
Enhancer region
binding site far
upstream of gene
turns transcription
on HIGH
Gives RNA Polymerase a
chance to “warm up”
Transcription Factors Initiation complex
transcription factors bind to promoter region
suite of proteins which bind to DNA
hormones?
turn on or off transcription
trigger the binding of RNA polymerase to DNA
Matching bases of DNA & RNA Match RNA bases to DNA bases on one of
the DNA strands
U
A G G G G G G T T A C A C T T T T T C C C C A A
U
U U
U
U
G
G
A
A
A C C RNA polymerase
C
C
C
C
C
G
G
G
G
A
A
A
A
A
5' 3'
Transcription: the process 1.Initiation~ transcription
factors mediate the binding of RNA polymerase to an initiation sequence (TATA box)
2.Elongation~ RNA polymerase continues unwinding DNA and adding nucleotides to the 3’ end (makes the mRNA strand)
3.Termination~ RNA polymerase reaches terminator sequence
Eukaryotic genes have junk!
Eukaryotic genes are not continuous
exons = the real gene
expressed / coding DNA
introns = the junk
inbetween sequence
eukaryotic DNA
exon = coding (expressed) sequence
intron = noncoding (inbetween) sequence
introns come out!
mRNA splicing
eukaryotic DNA
exon = coding (expressed) sequence
intron = noncoding (inbetween) sequence
primary mRNA transcript
mature mRNA transcript
pre-mRNA
spliced mRNA
Post-transcriptional processing eukaryotic mRNA needs work after transcription
primary transcript = pre-mRNA
mRNA splicing
edit out introns
make mature mRNA transcript
~10,000 bases
~1,000 bases
RNA Processing in Eukaryotes
5’ 3’
Modification of 5’ and 3’ ends
Pre-mRNA (hnRNA)
Spicing of exons
5’CAP Poly A tail Exon1 Intron1 Exon2 Intron2 Exon3 Intron3 Exon4
1977 | 1993
Richard Roberts Philip
Sharp CSHL
MIT adenovirus
common cold
Discovery of exons/introns
beta-thalassemia
Splicing must be accurate No room for mistakes!
a single base added or lost throws off the reading frame (mutation)
AUG|CGG|UCC|GAU|AAG|GGC|CAU
AUGCGGCTATGGGUCCGAUAAGGGCCAU AUGCGGUCCGAUAAGGGCCAU
AUG|CGG|GUC|CGA|UAA|GGG|CCA|U
AUGCGGCTATGGGUCCGAUAAGGGCCAU AUGCGGGUCCGAUAAGGGCCAU
Met|Arg|Ser|Asp|Lys|Gly|His
Met|Arg|Val|Arg|STOP|
RNA splicing enzymes
snRNPs
exon exon intron
snRNA
5' 3'
spliceosome
exon excised intron
5'
5'
3'
3'
3'
lariat
exon mature mRNA
5'
No, not smurfs! “snurps”
snRNPs
small nuclear RNA
proteins
Spliceosome
several snRNPs
recognize splice site
sequence
cut & paste gene
Alternative splicing Alternative mRNAs produced from same gene
Introns for one gene may be exons for another
different segments treated as exons
Starting to get hard to define a gene!
More post-transcriptional processing Need to protect mRNA on its trip from nucleus to cytoplasm
enzymes in cytoplasm attack mRNA protect the ends of the mRNA
add 5 GTP cap
add poly-A tail
longer tail, mRNA lasts longer: produces more protein
Translation
from
mRNA language
to
amino acid language
Players in Translation
mRNA – Code Ribosome – synthesizes protein tRNA – adaptor molecule, brings AA to ribosomes Amino acids Aminoacyl tRNA synthetases - attach amino acids to tRNAs
tRNA
Transfer RNA structure “Clover leaf” structure
anticodon on “clover leaf” end
amino acid attached on 3 end
Loading tRNA Aminoacyl tRNA synthetase
enzyme which bonds amino acid to tRNA
bond requires energy
ATP AMP
bond is unstable
so it can release amino acid at ribosome easily
activating enzyme
anticodon tRNATrp binds to UGG codon of mRNA
Trp Trp Trp
mRNA A C C U G G
C=O
OH OH
H2O O
tRNATrp
tryptophan attached to tRNATrp
C=O
O
Ribosomes Facilitate coupling of
tRNA anticodon to
mRNA codon
organelle or enzyme?
Structure
ribosomal RNA (rRNA) & proteins
2 subunits
large
small
E P A
Ribosomes
Met
5'
3'
U U A C
A G
A P E
A site (aminoacyl-tRNA site)
holds tRNA carrying next amino acid to be added to chain
P site (peptidyl-tRNA site)
holds tRNA carrying growing polypeptide chain
E site (exit site)
empty tRNA
leaves ribosome
from exit site
Ribosomes
How does mRNA code for proteins?
TACGCACATTTACGTACGCGG DNA
AUGCGUGUAAAUGCAUGCGCC mRNA
Met Arg Val Asn Ala Cys Ala protein
?
How can you code for 20 amino acids with only 4
nucleotide bases (A,U,G,C)?
4
4
20
ATCG
AUCG
AUGCGUGUAAAUGCAUGCGCC mRNA
mRNA codes for proteins in triplets
TACGCACATTTACGTACGCGG DNA
AUGCGUGUAAAUGCAUGCGCC mRNA
Met Arg Val Asn Ala Cys Ala protein
?
codon
Cracking the code 1960 | 1968
Crick
determined 3-letter (triplet) codon system
Nirenberg & Khorana
WHYDIDTHEREDBATEATTHEFATRAT WHYDIDTHEREDBATEATTHEFATRAT
Nirenberg (47) & Khorana (17)
determined mRNA–amino acid match
added fabricated mRNA to test tube of ribosomes, tRNA & amino acids
created artificial UUUUU… mRNA
found that UUU coded for phenylalanine
1960 | 1968 Marshall Nirenberg
Har Khorana
The code Code for ALL life!
strongest support for a
common origin for all life
Code is redundant
several codons for each amino
acid
3rd base “wobble”
Start codon
AUG
methionine
Stop codons
UGA, UAA, UAG
Why is the wobble good?
How are the codons matched to
amino acids?
TACGCACATTTACGTACGCGG DNA
AUGCGUGUAAAUGCAUGCGCC mRNA
amino acid
tRNA anti-codon
codon
5 3
3 5
3 5
UAC
Met
GCA
Arg
CAU
Val
Building a polypeptide Initiation
brings together mRNA, ribosome subunits, initiator tRNA
Elongation adding amino acids based on codon sequence
Translocation – Ribosome ratchets over on codon. The tRNA that was in the A site is moved to the P site. The uncharged tRNA in the P site exits the ribosome through the E site.
Termination end codon
When ribosome reaches the stop codon a release factor binds to the A site and triggers the release of the polypeptide. The ribosome releases the tRNA and the mRNA.
1 2 3
Leu
Leu Leu Leu
tRNA
Met Met Met Met
P E A
mRNA 5' 5' 5' 5'
3' 3' 3' 3'
U U A A A A C
C C
A U U G G G U
U A
A A A C
C C
A U U G G G U
U A
A A A C
C C
A U U G G G U U
A A A C
C A U U G G
Val Ser
Ala Trp
release factor
A A A
C C U U G G 3'
Fig. 17-18-4
Amino end of polypeptide
mRNA
5
3 E
P site
A site
GTP
GDP
E
P A
E
P A
GDP
GTP
Ribosome ready for next aminoacyl tRNA
E
P A
The Functional and Evolutionary Importance of Introns
• Some genes can encode more than one kind of polypeptide, depending on which segments are treated as exons during RNA splicing
• Such variations are called alternative RNA splicing
• Because of alternative splicing, the number of different proteins an organism can produce is much greater than its number of genes
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Fig. 17-12
Gene DNA
Exon 1 Exon 2 Exon 3 Intron Intron
Transcription
RNA processing
Translation
Domain 2
Domain 3
Domain 1
Polypeptide
Polysomes – teamed ribosomes translating together
• Polypeptide synthesis always begins in the cytosol (cytoplasm)
• Synthesis finishes in the cytosol unless the polypeptide signals the ribosome to attach to the ER
• Polypeptides destined for the ER or for secretion are marked by a signal peptide
• A signal-recognition particle (SRP) binds to the signal peptide
• The SRP brings the signal peptide and its ribosome to the ER
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Proteins targeted to ER
Can you tell the story?
DNA
pre-mRNA
ribosome
tRNA
amino acids
polypeptide
mature mRNA
5' GTP cap
poly-A tail large ribosomal
subunit
small ribosomal subunit
aminoacyl tRNA synthetase
E P A
5'
3'
RNA polymerase
exon intron
tRNA
END Protein Synthesis
Prokaryote vs. Eukaryote genes Prokaryotes
DNA in cytoplasm
circular chromosome
naked DNA
no introns
Eukaryotes
DNA in nucleus
linear chromosomes
DNA wound on histone
proteins
introns vs. exons
eukaryotic DNA
exon = coding (expressed) sequence
intron = noncoding (inbetween) sequence
introns come out!
Transcription & translation are simultaneous in bacteria
DNA is in
cytoplasm
no mRNA
editing
ribosomes
read mRNA
as it is being
transcribed
Translation in Prokaryotes
Translation: prokaryotes vs. eukaryotes Differences between prokaryotes & eukaryotes
time & physical separation between processes takes eukaryote ~1 hour
from DNA to protein
no RNA processing
When do mutations affect the next generation?
Mutations Point mutations
single base change
base-pair substitution silent mutation
no amino acid change
redundancy in code
missense
change amino acid
nonsense
change to stop codon
Point mutation leads to Sickle cell anemia What kind of mutation?
Missense!
Sickle cell anemia Primarily in African races/descendants
recessive inheritance pattern
strikes 1 out of 400 African Americans
hydrophilic amino acid
hydrophobic amino acid
Mutations Frameshift
shift in the reading frame changes everything “downstream”
insertions adding base(s)
deletions losing base(s)
Where would this mutation cause the most change: beginning or end of gene?
Cystic fibrosis Primarily European races/descendants
strikes 1 in 2500 births
1 in 25 whites is a carrier (Aa)
normal allele codes for a membrane protein
that transports Cl- across cell membrane
defective or absent channels limit transport of Cl- (& H2O) across cell
membrane
thicker & stickier mucus coats around cells
mucus build-up in the pancreas, lungs, digestive tract & causes bacterial
infections
without treatment children die before 5;
with treatment can live past their late 20s
Deletion leads to Cystic fibrosis
loss of one amino acid
delta F508
2007-2008
What’s the value of mutations?