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 53
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