Unit 6 Molecular Biology

Fridrich Meischer

• First to discover DNA. 

• Extracted white blood cells from blood and lysed the cells. 

• Called it ‘Nuclein’. (later to be called nucleic acids)

Phoebus Levene

• Determined nucleotide structure: Base, Sugar, Phosphate Group

• Proposed the tetranucleotide hypothesis, which claimed DNA was a simple, repeating sequence and therefore too uniform to carry genetic information. That proved to be incorrect)

Griffith Experiment

• Griffith was working with bacteria that caused pneumonia. 

• S (Smooth) strain of bacteria would kill the mouse. 

• R (Rough) strain would not kill the mouse.   

• Boiled S strain Injected boiled S strain into the mouse and the mouse was ok!

• Mixed boiled S strain and R strain and injected it into the mouse. Killed the mouse. 

    -> He found living S strain in the dead mice

Came up with Transformation Principle.

Transformation Principle

Griffith Experiment

• Bacteria can transfer genetic material through transformation.

Beadle and Tatum

Showed that genes control the production of enzymes.

• Used bread mold (Neurospora crassa).

• X-rays were used to create mutations.

• Mutant molds could no longer make certain molecules needed to grow.

• When a missing nutrient was added back, growth resumed.

Conclusion:

Each gene is responsible for making one enzyme (later refined to one gene → one polypeptide).

• One gene-one enzyme hypothesis

Avery, McLeod, McCarty

• Built on the work of Griffith to show that DNA (not protein) was the transforming principle

THE PROCESS:

• Isolated DNA, RNA, and proteins from heat-killed virulent S. pneumoniae bacteria and tested which ones could transform non-virulent R-strain bacteria.

• Only DNA was able to transform the non-virulent bacteria into the virulent form.

• Degradation Tests: When DNA was treated with DNA-digesting enzymes (DNase), transformation did not occur, but treatments with protease (breaking down proteins) or RNase (breaking down RNA) transformation occured.

Central Dogma

DNA is transcribed into RNA and translated into protein

• DNA —> RNA —> Protein

Hershey and Chase

• Waring Blender Experiment

1. Two groups of bacteriophages were labeled:

• 35S (sulfur) labeled the protein coat because sulfur is in proteins, not DNA.

• 32P (phosphorus) labeled the DNA because phosphorus is in DNA, not proteins.

2. The bacteriophages infected bacteria.

3. A blender separated the viral coats from the bacteria.

4. Centrifugation separated the parts by density.

5. The bacteria contained 32P, showing that DNA entered the cells, not protein.

James Watson and Francis Crick

• Proposed the double-helix structure of DNA in 1953

• Showed that DNA consists of two antiparallel strands with complementary base pairing (A–T and C–G)

• Heavily relied on Rosalind Franklin

Maurice Wilkins

• Who “shared” Franklin’s photo with James Watson and Francis Crick. 

• Watson and Crick created the 1st accurate model of DNA with Franklin’s work. 

Rosalind Franklin

• Scientists knew DNA was made of nucleotides, but didn’t understand total structure. 

• Studied x-ray diffraction and was studying DNA. 

  • Took “Photo 51”

Linus Pauling

• Alpha-Helix Structure: In the 1950s, Pauling proposed the alpha-helix as a key structural feature of proteins, a breakthrough that helped understand protein folding. He also was the part of the discovery of β sheets.

• Protein Structure: He emphasized the role of hydrogen bonds in protein stability w/in secondary structure.

• DNA Structure Hypothesis: Pauling attempted to model the structure of DNA, proposing a triple-helix model in 1953, which was later shown to be incorrect.

DNA Structure

• The monomers of DNA are nucleotides.

• Nucleotides are made of base+sugar+phosphate. Phosphate Group, PO₄^3-, deoxyribose sugar, Nitrogenous base: purine or pyrimidine

Eukaryotic Chromosome

Shape - Linear

Size - Large

Number - Multiple

Location - Nucleus

Storage proteins - Histones

Prokaryotic Chromosome

Shape - Circular

Size - Small

Number - Single

Location - Nucleoid (region in cytoplasm)

Storage proteins - Nucleoid associated proteins (supercoiling)

Double Bonds

A and T

Triple Bonds

G and C

DNA Bases

A, G, T, C

RNA Bases

A, G, U, C

Pyrimidines

Single Rings

• Cytosine

• Thymine (DNA only)

• Uracil (RNA only)

Purines

Double Rings

• Guanine

• Adenine

Bases Bonding

• Sugar-phosphate backbone

• Bases bound with hydrogen bonds

• Pyrimidines bound to purines

Chargaff’s Rule

• % A = % T

• % G = % C

• He figured out base pairing

• This was crucial information that helped Watson & Crick

• This is true for all species 

Double Helix

Two chains of nucleotides in a twisted ladder structure called a

Phosphodiester Bonds (covalent)

Backbone is composed of repeating deoxyribose sugar and phosphate bonded connected by

Hydrogen Bonds

Nitrogenous bases form the rungs of the ladder and are connected by

Antiparallel

• DNA runs 5’ to 3’ and 3’ to 5’.

• One end is 5’ and the other 3’. 

  • 5’ end is phosphate

→ 5’ PO₄^3-

• 3’ end is sugar

  → 3’OH^–

Major and Minor Grooves

The two unequal spaces that run along the outside of the DNA double helix.

• Major Groove - Larger

• Minor Groove - Smaller

Chromosome

Tightly packed DNA and protein structure. DNA takes on this form as the cell prepares to undergo division. (“butterfly”)

Histone

Protein molecule that DNA wraps itself around. (Spools)

• Positively charged because they contain a lot of arginine and lysine, both of which carry a net positive charge

• Then bind to negatively charged phosphate groups in the sugar phosphate backbone of DNA

Chromatin

Thin thread of DNA. Consists of DNA and histones. It will condense before mitosis. (“spaghetti”)

Phosphate groups (charged)

Negatively charged

8 Histones

DNA coils around

Nucleosomes

DNA is coiled around 8 histone proteins to form

Chromatin (form)

Nucleosomes are coiled again to form

DNA Packaging Steps

1. DNA wraps around histone proteins forming beads on string” called nucleosomes. 

2. Nucleosomes further coil and condense/gather to form chromatin. 

3. Chromatin fibers can unwind for DNA replication and transcription.

Plasmids

• Small, circular pieces of double-stranded DNA found in prokaryotes and some eukaryotes.

• Often carry helpful genes like antibiotic resistance.

• Used as vectors to carry new genes into cells for gene cloning, genetic engineering (such as making insulin), and gene therapy.

Conservation (Replication)

The parent double-helix DNA is copied in its entirety, and the new cell’s DNA is entirely a copy of the old.

Dispersive (Replication)

DNA is chopped up into pieces; these little pieces are copied and then reassembled in combination with the old pieces.

Semiconservative (Replication)

• DNA Replication is

The double-stranded DNA separate from their helix shape, and each makes a copy of itself. The new cells then contain one strand from the parent cell and one newly synthesized strand.

Meselson & Stahl Experiment

“The Most Beautiful Experiment in Molecular Biology” 

How did we know the DNA Replication is semiconservative

Meselson & Stahl

1. Bacteria were first grown in ¹⁵N (heavy nitrogen) so their DNA became heavy.

2. A sample of this ¹⁵N DNA was saved.

3. The rest were moved to ¹⁴N (light nitrogen) so new DNA would be light.

4. Samples were taken after each time the bacteria doubled.

5. A sample grown only in ¹⁴N was used for comparison.

6. DNA was taken out and mixed with a strong salt solution.

7. The samples were spun fast in a centrifuge.

8. Heavy DNA (¹⁵N) sank lower than light DNA (¹⁴N), showing differences in DNA.

S Phase

DNA replication occurs in

1. Initiation

Origin of replication: Specific DNA sequences are recognized by initiator proteins.

• Helicase unwinds the DNA: Unzips the double helix, creating a replication bubble with two replication forks.

2. Primer Synthesis

• Primase synthesizes short RNA primers on the single-stranded DNA templates to provide a starting point for DNA synthesis.

3. Elongation

• DNA polymerase III adds nucleotides to the 3' end of the primer, synthesizing the new DNA strand in the 5' to 3' direction.

• On the leading strand, synthesis is continuous. On the lagging strand, DNA is synthesized in Okazaki fragments.

4. Primer Removal

• DNA polymerase I removes RNA primers and fills in the gaps with DNA.

5. Ligation

• DNA ligase seals the nicks between adjacent DNA fragments, joining the newly synthesized DNA strands into a continuous strand.

Origin of Replication

• Location where replication begins. This is where the unzipping begins.

  • Euk: multiple (b/c large genome)

  • Prok: single

Replication Fork

Y-shaped area where DNA is being copied. The enzyme helicase unwinds the double-stranded DNA, creating the fork so new DNA strands can be made.

Helicase

• Unwinds DNA by breaking hydrogen bonds between bases

• Requires ATP (ATP hydrolysis)

Topisomerase

Relieves torsional strain/supercoiling caused by unwinding

Single stranded binding proteins

• Binds to ssDNA to prevent it from reannealing (coming back to double strands)

• Stabilize strands to keep them single-stranded, no enzymatic activity

Primase

• Enzyme that moves 5’ to the 3’

• Lays down RNA primers by reading ssDNA

• Creates short RNA sequence (about 10 to 15 nucleotides long)

Primers

Necessary because DNA Pol III needs a free 3'OH to add nucleotides to the growing chain.

DNA Polymerase 3

• Enzyme that binds RNA primers and adds nucleotides to elongate the DNA strand in the 5’ to 3’ direction.

• It forms a phosphodiester bond by joining the 3' OH of the growing DNA strand to the 5' phosphate of the new nucleotide.

• VERY ACCURATE

DNA Polymerase 1

Replaces RNA primers with DNA and proofreading

Ligase

• Seals nicks between DNA fragments

• Forms phosphodiester bonds between the sugar-phosphate backbones of adjacent nucleotides. Requires ATP.

Leading strand

• Made continuously, DNA Pol III moves towards replication fork. 

• 5’ to 3’ direction

Lagging strand

• Made discontinuously, DNA Pol III moves away from replication fork. Creates small sections known as Okazaki fragments

• 3’ to 5’ direction

3’ to 5’

DNA polymerase can only read and add nucelotides to the template of

5’ to 3’

The new strand grows

5' to 3' Exonuclease Activity (DNA Poly 1)

Removes RNA primers. removes RNA primers by cleaving the RNA bases in the 5' to 3' direction

3' to 5' Exonuclease Activity (DNA Poly 1)

• Proofreading function, moves backwards along the newly synthesized DNA (in the 3' to 5' direction)

• Removes nucleotides one at a time from the end of a DNA strand, fixing mistakes during DNA replication

Endonuclease activity

Cuts nucleotides in the middle of a strand rather than at the ends, can cut out damaged or mismatched DNA 

Methylation (CH3)

DNA is after it’s replicated. 

• Happens to control gene activity. It can turn genes off or down, helping the cell know which genes to use and which not to use.

- Prok: usually A is methylated

- Euk: usually C is methylated

Excision Repair

Fixes damaged DNA, like damage from UV light or chemicals (thymine dimers). The damaged section is cut out and replaced with correct DNA.

Mismatch Repair

• Happens after DNA replication.

• The cell finds and fixes mistakes that were missed by proofreading by cutting out a section of the new strand and replacing it.

Xeroderma pigmentosum

• Rare Genetic Disorder

• Defective Excision Repair because of a mutation in one of the genes responsible for excision repair.

• Recall: UV radiation from sun can cause Thymine-Thymine dimers. 

• Can’t go out in sunlight

• Increased skin cancers/cataracts

• 1 in 1,000,000 million