Download Structure and Replication of DNA

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
Phage
head
Tail sheath
Tail fiber
Bacterial
cell
100 nm
DNA
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
Humans
Chicken
3.25pg
1.267pg
Somatic Cells
7.30 pg
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 Structure –
Chromatin = unwound DNA
Nucleosome
(10 nm in diameter)
DNA
helix
in diameter)
double
(2 nm
H1
Histones
DNA, the double helix
video
Histones
Histone tail
Nucleosomes, or “beads on
a string” (10-nm fiber)
Chromatin coils around proteins to form Chromosomes
Chromatid
(700 nm)
30-nm fiber
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)
15N
DNA
One Cell Division
in 14N
15N/14N
DNA
2nd Cell Division
in 14N
14N
DNA
15N/14N
DNA
Less Dense
More Dense
Density Centrifugation
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
Video
Origins of Replication
Fig. 16-13
Primase
Single-strand binding
proteins
3
Topoisomerase
5
3
5
Helicase
5
RNA
primer
3
DNA Polymerase – enzyme that builds the new strand
5’
3’
3’
Pol
5’
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
3’
5’
Pol
Leading Strand
Lagging Strand
Pol
3’
RNA
Primer
5’
Video
5’
3’
Other Proteins at Replication Fork
3’
5’
DNA Pol III
Single Stranded
Binding Proteins
Pol
Leading Strand
DNA Pol I
Ligase
Lagging Strand
Pol
Helicase
3’
5’
Primase
5’
3’
Lagging strand
assembly and
Okazaki
fragments
Overview
Origin of replication
Lagging strand
Leading strand
Lagging strand
2
1
Leading strand
Overall directions
of replication
3
5
5
Template
strand
3
RNA primer
3
5
3
1
5
3
5
Okazaki
fragment
3
1
5
3
5
2
3
5
2
3
3
5
1
3
5
1
5
2
1
3
5
Overall direction of replication
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
5
Ends of parental
DNA strands
Leading strand
Lagging strand
3
Last fragment
Previous fragment
RNA primer
Lagging strand
5
3
Parental strand
Removal of primers and
replacement with DNA
where a 3 end is available
5
3
Second round
of replication
5
New leading strand 3
New lagging strand 5
3
Further rounds
of replication
Shorter and shorter daughter molecules
• 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?
DNA
replication
RNA
protein
DNA gets
all the glory,
but proteins do
all the work!
trait
a
a
From gene to protein
nucleus
DNA
cytoplasm
transcription
mRNA
a
a
translation
ribosome
a
a
a
a
a
a
a
a
a
a
a
a
protein
a
a
a
a
a
a
trait
Genetic Code
5’
Identifying Polypeptide Sequence
GACGACGGAUGCGCAAUGCGUCUCUAUGAGACGUAGCUCAC
• 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]
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…
DNA
transcription
RNA
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
5
DNA
C
G
3
build RNA 53
A
G
T
A T C
T A
rewinding
mRNA 5
coding strand
G
C
A G C
A
T
C G T
T
A
3
G C A U C G U
C
G T A G C A
T
A
T
RNA polymerase
C
A G
C T
G
A
T
A
T
3
5
unwinding
template strand
Animation of Transcription
• http://vcell.ndsu.nodak.edu/animations/trans
cription/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
G
the DNA strands
G
U
C
A
A G
C
A
U
G
U
A
C
G
A
U
A
C
5'
RNA
A C C polymerase G
A
U
3'
T G G T A C A G C T A G T C A T C G T A C C G T
U
C
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
introns
come out!
intron = noncoding (inbetween) sequence
eukaryotic DNA
exon = coding (expressed) sequence
mRNA splicing
 Post-transcriptional processing
 eukaryotic mRNA needs work after transcription
 primary transcript = pre-mRNA
 mRNA splicing
 edit out introns
 make mature mRNA transcript
intron = noncoding (inbetween) sequence
~10,000 base
eukaryotic DNA
exon = coding (expressed) sequence
primary mRNA
transcript
mature mRNA
transcript
pre-mRNA
~1,000 base
spliced mRNA
RNA Processing in Eukaryotes
Pre-mRNA (hnRNA)
5’
3’
Modification of 5’ and 3’ ends
5’CAP
Exon1
Intron1 Exon2
Intron2
Exon3
Intron3 Exon4
Spicing of exons
Poly A tail
1977 | 1993
Discovery of exons/introns
Richard
Roberts
CSHL
Philip
Sharp
MIT
beta-thalassemia
adenovirus
common cold
Splicing must be accurate
 No room for mistakes!
 a single base added or lost throws off the reading frame (mutation)
AUGCGGCTATGGGUCCGAUAAGGGCCAU
AUGCGGUCCGAUAAGGGCCAU
AUG|CGG|UCC|GAU|AAG|GGC|CAU
Met|Arg|Ser|Asp|Lys|Gly|His
AUGCGGCTATGGGUCCGAUAAGGGCCAU
AUGCGGGUCCGAUAAGGGCCAU
AUG|CGG|GUC|CGA|UAA|GGG|CCA|U
Met|Arg|Val|Arg|STOP|
RNA splicing enzymes
 snRNPs
 small nuclear RNA
 proteins
exon
 Spliceosome
5'
snRNPs
snRNA
intron
exon
3'
 several snRNPs
 recognize splice site
sequence
 cut & paste gene
No,
not smurfs!
“snurps”
spliceosome
5'
3'
lariat
5'
exon
mature mRNA
5'
3'
exon
3'
excised
intron
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
Trp
activating
enzyme
C=O
OH
OH
Trp
C=O
O
Trp
H 2O
tRNATrp
anticodon tryptophan
attached to tRNATrp
O
AC C
UGG
mRNA
tRNATrp binds to UGG
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
 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
Met
U A C
A U G
5'
E
P
A
3'
Ribosomes
How does mRNA code for proteins?
DNA
TACGCACATTTACGTACGCGG
4 ATCG
mRNA
4 AUCG
AUGCGUGUAAAUGCAUGCGCC
protein
?
Met Arg Val Asn Ala Cys Ala
20
How can you code for 20 amino acids with only 4
nucleotide bases (A,U,G,C)?
mRNA codes for proteins in triplets
DNA
TACGCACATTTACGTACGCGG
codon
mRNA
AUGCGUGUAAAUGCAUGCGC
?
protein
Met Arg Val Asn Ala Cys Ala
Cracking the code
1960 | 1968
Nirenberg & Khorana
 Crick
 determined 3-letter (triplet) codon system
WHYDIDTHEREDBATEATTHEFATRAT
WHYDIDTHEREDBATEATTHEFATRA

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
Marshall Nirenberg
1960 | 1968
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”
Why is the
wobble good?

Start codon



AUG
methionine
Stop codons

UGA, UAA, UAG
How are the codons matched to
amino acids?
DNA
mRNA
3
5
5
3
TACGCACATTTACGTACGCGG
AUGCGUGUAAAUGCAUGCGC
3
tRNA
amino
acid
codon
5
UAC
GCA
anti-codon
CAU
Met
Arg
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.
3 2 1
Val
Leu
Met
Met
Met Leu
Met
Leu
Ala
Leu
release
factor
Ser
Trp
tRNA
UAC
5'
C UG A A U
mRNA A U G
3'
E
P A
5'
UA C G A C
A U G C U GA A U
5'
3'
U A C GA C
A U G C UG AA U
3'
5'
U AC G A C AA U
A U G C UG
3'
A CC
U GG UA A
3'
Fig. 17-18-4
Amino end
of polypeptide
E
3
mRNA
Ribosome ready for
next aminoacyl tRNA
P
A
site site
5
GTP
GDP
E
E
P A
P A
GDP
GTP
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
Intron
Exon 2
Intron
Exon 3
Transcription
RNA processing
Translation
Domain 3
Domain 2
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
RNA polymerase
DNA
Can you tell
the story?
amino
acids
exon
pre-mRNA
intron
5' GTP cap
mature mRNA
large ribosomal
subunit
5'
small ribosomal
subunit
tRNA
poly-A tail
aminoacyl tRNA
synthetase
3'
polypeptide
tRNA
E P A
ribosome
END Protein Synthesis
Prokaryote vs. Eukaryote genes
 Prokaryotes
 Eukaryotes
 DNA in cytoplasm
 DNA in nucleus
 circular chromosome
 linear chromosomes
 naked DNA
 DNA wound on histone
 no introns
proteins
 introns vs. exons
introns
come out!
intron = noncoding (inbetween) sequence
eukaryotic
DNA
exon = coding (expressed) sequence
Translation in Prokaryotes
 Transcription & translation are simultaneous in bacteria
 DNA is in
cytoplasm
 no mRNA
editing
 ribosomes
read mRNA
as it is being
transcribed
Translation: prokaryotes vs. eukaryotes
 Differences between prokaryotes & eukaryotes
 time & physical separation between processes
 takes eukaryote ~1 hour
from DNA to protein
 no RNA processing
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
When do mutations
affect the next
generation?
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
delta F508
loss of one
amino acid
What’s the value of
mutations?
2007-2008