Structure and Replication of DNA...DNA Replication: Semiconservative Replication- DNA unzips and a new strand builds on the inside. The new strands each have a piece of the “old”

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

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

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

DNA, the double helix Histones Nucleosomes, or “beads on a string” (10-nm fiber)

DNA Structure – Chromatin = unwound DNA

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

• 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

Origins of Replication Video

Fig. 16-13

Topoisomerase

Helicase

Primase Single-strand binding

proteins

primer

DNA Polymerase – enzyme that builds the new strand

3’ 5’

3’ 5’ 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

Leading Strand

Lagging Strand

RNA Primer

Other Proteins at Replication Fork

Leading Strand

Lagging Strand

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

Lagging strand

Overall directions

of replication

Template

strand

RNA primer

Okazaki

fragment

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

Replicating Ends of Linear Chromosomes

Fig. 16-19

Ends of parental

DNA strands Leading 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

• 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

Telomerase

Telomeres

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!

From gene to protein

DNA transcription

nucleus cytoplasm

protein translation

ribosome

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

DNA nucleic acid language

RNA nucleic acid language

RNA ribose sugar

N-bases

uracil instead of thymine

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

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

A G G G G G G T T A C A C T T T T T C C C C A A

A C C RNA polymerase

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

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

spliceosome

exon excised intron

lariat

exon mature mRNA

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

mRNA language

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

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

tRNATrp

tryptophan attached to tRNATrp

Ribosomes Facilitate coupling of

tRNA anticodon to

mRNA codon

organelle or enzyme?

Structure

ribosomal RNA (rRNA) & proteins

2 subunits

Ribosomes

U U A C

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)?

mRNA codes for proteins in triplets

Met Arg Val Asn Ala Cys Ala protein

Cracking the code 1960 | 1968

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

3rd base “wobble”

Start codon

methionine

Stop codons

UGA, UAA, UAG

Why is the wobble good?

How are the codons matched to

amino acids?

amino acid

tRNA anti-codon

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.

Leu Leu Leu

Met Met Met Met

mRNA 5' 5' 5' 5'

3' 3' 3' 3'

U U A A A A C

A U U G G G U

A A A C

A U U G G G U

A A A 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

C C U U G G 3'

Fig. 17-18-4

Amino end of polypeptide

P site

A site

Ribosome ready for next aminoacyl tRNA

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

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

Proteins targeted to ER

Can you tell the story?

pre-mRNA

ribosome

amino acids

polypeptide

mature mRNA

5' GTP cap

poly-A tail large ribosomal

subunit

small ribosomal subunit

aminoacyl tRNA synthetase

RNA polymerase

exon intron

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

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?

Structure and Replication of DNA...DNA Replication: Semiconservative Replication- DNA unzips and a new strand builds on the inside. The new strands each have a piece of the “old”

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