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1/10/2020 - “Once information can get into a protein, it

can’t get out again”
Molecular Biology and Biotechnology
● The molecular basis for biological Biomolecules involved:
phenomenon 1. DNA (ATGCs)
● Cellular processes - base sequence
● Metabolic reactions 2. Protein
● Molecular structures and functions - Amino acid sequence
○ Molecules act as machines - Becomes a 3D structure (tertiary)
(Structural biology)
○ How structure affects function How do Amino Acids become 3D structures?
● Understanding key concepts before - The character of the amino acid sequence
applications and innovations tells about the type of intermolecular forces
● Draws elements from: physics, chemistry, between them
biology - Kind (among 20) governs the interactions
and thus the shape of the 3D structure
Central Dogma - Ex. disulfide bridges, alpha helices, etc.
- Involves: DNA, RNA, proteins
- Process: transcription, translation, Information transfer:
replication, ​reverse transcription - DNA -> DNA (replication)
- Crick: demonstrates the transfer of genetic - DNA -> RNA (transcription: 1st step of
information nucleosynthesis)
- ATGCs (bases) -> Amino Acid sequence -> - RNA -> protein (translation)
Tertiary/3D structure
Reverse Transcription (1975)
- Temin & Baltimore
- useful in cloning

“Impossible” Transfers
- Protein -> protein
- Protein -> RNA
- Protein -> DNA

Prions
- Protein viruses
- Mad cow disease
What is a Dogma? - Scrapie studies
- Something held as an established opinion; - Infected affects non-infected
authoritative tenet proteins
- Not necessarily the truth - Misfolding -> aggregates -> plaques
- Doctrine or body of doctrines concerning - Change in a shape of an existing protein
faith or morals formally stated/proclaimed - Chaperonins -> can correct misfolding
by the church
- Origin: Dokein (Greek) -> to seem 1/15/2020
good/think it is good
Central Dogma
The Central Dogma (1957) - Transfer of genetic information
- Was originally a proposal -> ​Francis Crick - Not unidirectional due to reverse
- DNA passed on genetic information transcription
- Why undergo reverse transcription?
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a. Viruses: change RNA to DNA Other observations:
- Hijack mechanism of - 34 A and 3.4 A were the different
bacteria to replicate DNA separations of repeating units
b. Cloning - Close together in x-ray diffraction -> actually
- DNA: more stable form of genetic far apart
information storage
- Seed DNA in expression vectors Types of Helices
- No splicing (unlike RNA) - 3 kinds: A, B, Z

Photo 51: Rosalind Franklin

- characteristic patterns which are unique to - Tall vs. squat

certain shapes
- x-ray ​fiber diffraction pattern
- Diffraction​: pass electromagnetic waves
through a material to generate scatter
waves into a pattern
- X pattern → ​HELIX
- Peak distances
- 3.4 A dist. bet. nitrogenous bases
- 34 A dist. of one turn
- Therefore, 10 base pairs comprise 1
turn of the helix
DNA Double Helix Structure (1953)
- Features:
1. Double stranded helix
2. Uniform dimensions
3. Complementary base pairs (A-T;
G-C)
- Implication:
- 2 strands could unzip
- Complementary base pairing meant
it was easy to determine base on the
other side
- one strand could serve as a
template or blueprint for copying
- Logical explanation for
TRANSCRIPTION and
REPLICATION

Genetics
- Branch of biology that deals with heredity
and variation of organisms
- From Greek: Genesis (origin)

Gregor Mendel
- Laws of Heredity (1865)
1. Segregation
2. Independent Assortment
3. Dominance

- Same laws apply in molecular biology ->
whether pathologic or non-pathologic
Mendelian Genetics
1) Law of Dominance
a) Dominant vs. recessive
b) Homozygous vs. heterozygous
c) Phenotype vs. genotype
DNA Material: Classic Experiments
A. Thomas Hunt Morgan (1915)
- Linear arrangement of genes
- Mechanism of Mendelian inheritance
- Fly: drysophilla
- Eye-color (white vs. red); X-linked
genetic trait
B. Barbara McClintock (1929)
- Genes exist as physical locations on
chromosomes
- Color or corn kernels on a cob
- Dominant alleles:
- Dominant - yellow phenotype
- Recessive - color dictated by the
other types of alleles present
- “Jumping genes”
- What disrupts
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- Transposable elements (genes) can
be disrupted, destroying its
functionality
C. Beadle and Tatum (1941)
- One gene, one enzyme hypothesis
- Fungi -> hit w/ x-rays -> cultured (complete
vs. minimal medium)
- Some genes are disrupted, w/c destroys its
function (mutation)
- Ex. cannot metabolize anymore
D. Griffith (1928)
- Transformation of pneumococcus
- heat-killed S strain transformed R into live S
strain
E. Avery, McCarty, McLeod (1944)
- DNAse disrupts transformation of
pneumococcus
- Non-transformation when DNAse was
disrupted
F. Hershey-Chase
- Bacteriophage transfer DNA to their
progeny
- Radioactive labelling:
- Sulfur (protein)
- Phosphorus (DNA)
G. Nirenberg (1965)
- Decoding the genetic code
- System:
- Cell free extract (cytoplasmic
components)
- Synthetic (RNA)
- Amino acid components
(Methionine, … , Trypsine)
- Individual radioactive amino acids
- The genetic code: mRNA transcript codes
for specific polypeptides; types of codons of
20 amino acids
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1/17/2020 Deoxyribonucleic Acid

Central Dogma (Review)

- The central dogma involves the transfer of
information NOT genetic material
- Replication: copying the same material
- Will not apply to the DNA -> RNA
transformation of info
- Use “transcribe”

DNA (As genetic material)

Discovery
A) Friendrich Miescher, MD (1868)
- Extracted a “novel molecule” from
lymphocyte nuclei/white blood cells
(Nuclein)
- Present in other cell nuclei
- Increases in amt. prior to cell division
Characteristics:
- Contained phosphorous, nitrogen
- Did not contain sulfur
- Nuclein was resistant to protease action

Isolation and Identification

B) Albrecht Kossel, MD (1885)
- Isolated “acidic components” from
Nuclein
- Identified 5 Nucleobases (Nitrogen)
- Guanine
- Adenine
- Cytosine
- Thymine
- Uracil
- Early work on proteins (1895)
- Nobel prize in physiology or
medicine (1910) - “knowledge of cell
chemistry made through his work on
proteins, including the nucleic
substances”
Diffraction Studies
C) Astbury & Bell (1938)
- X-ray diffraction studies
- 3.5 A repeating units which stacked
up
- “pile of pennies”
- single-stranded fiber with
nucleotides stacked one on top of
another perpendicular to the DNA
backbone
D) Chargaff’s Rules
- Human Deosoxypentose Nucleic
Acid: Composition
1. Complementary base pairing: A = T;
G=C
- Ratio of A = T; Ratio of G =
C
- Ratios of purines and
pyrimidines
- Approaches 1.0
2. Varying ratios of As and Ts and Gs
and Cs between species
- This manifests as diff.
genetic material or different
traits
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DNA Structure
E) Watson and Crick (1953)
- Features:
- Double stranded helix
- Uniform dimensions
- Complementary base pairing (A-T;
G-C)
Form A
- More compact form of DNA
- Dehydrated
- Base pairs follow the helix
- Deep major groove
- Occurs in DNA-RNA duplexes
- DNA capsules; thermophilic

Form B
- Ideal or model form of DNA Life Story: the Race for the Double Helix (1987)
- Bases paired horizontally (ladder)
- Hydrated form 1) James Watson
- Working with bacteriophage (1951)
Form Z - Phage group
- Extended structure/helix References:
- Left handed orientation a. Salvador Luna -> L or LB Broth; B=Bertani
- Interacts with B-DNA for modulating - Worked on bacteriophage studies
supercoils - Effect of host genotype on phage
phenotype (evolutionary)
Reasons: b. Max Belbruck
- Spacing has to do with the difference - Studied the replication of viruses
between the nitrogenous bases
● Form A: 2.7 A 2) Maurice Wilkins
● Form B: 3.4 A - Fiber diffraction techniques for DNA
- causes -> proteins bind with them to shift (specialized camera)
their confirmation - Thin crystal then shoot light ray across to
produce images

3) Rosalind Franklin
- Fiber diffraction of form A and form B types
of DNA

4) Francis Crick
- Lecture with which the central dogma is
based

5) John Turton Randall

- Related DNA components w/ air within
cameras
- hydrogen saturation may provide more
details
- Physicist → sharp images in a micrograph

​ A-DNA ​ ​ B-DNA​ ​Z-DNA

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6) Raymond Gosling ● General Theory: genetic material in
- Crystallographer that worked w/ Rosalind primordial organisms started with RNA
Franklin instead of DNA
- Graduate student ● How do the biomolecules involved perform
- Structure published: Gosling and Franklin the said function?
○ Based on their structures; structural
7) Sir William Lawrence Bragg biology
- Nobel Prize Winner
- X-ray diffraction studies: Bragg’s Law Biomolecule Structure and Functions
- Angle differences → how we determine ● Replication Process
different structures - Interaction of the DNA and proteins,
specifically DNA/RNA polymerase
8) Max Perutz - Begins with RNA primase w/c
- 3D structure of hemoglobin synthesizes short RNA sequences
- Diffracting phasing by heavy atom called primers
replacement - Proteins clamp onto the DNA strand

9) John Kendrew
- Structure of myoglobin

10) Erwin Chargaff

- A-T; C-G
- Certain ratio of A&T and G&C
- DNA composition does not change as it
ages
- Different ratio for diff organisms/species - DNA in one strand makes it more
accessible to proteins
2/5/2020 ● Transcription/Reverse Transcription
RNA - Central Dogma (1957) - DNA: template/product
Processes DNA RNA Protein
- RNA: product/template
Involvement Involvement Involvement - DNA/RNA polymerase, reverse
transcriptase to
Replication YES YES YES
- serves as - primer - DNA polymerase ● Translation
template and (primase) - helicase
product (polymerization of
- Ribosomes:
nucleotides) - made of proteins and rRNA
- Has 2 parts: small and large
Reverse YES YES YES
Transcription - expected - template - reverse subunit
product from transcriptase
RNA (cRNA) - DNA polymerase

Transcription YES YES YES

- template for - product - RNA polymerase
RNA transcript

Translation NO YES YES

- template - product
(mRNA) - ribosomal
- ribosomes proteins
(rRNA)
- transfer
(tRNA): binds
with the
ribosome

- 3D render or S & L subunits:
- Orange: coiled structure (helices,
probably rRNA)
→ not hollow; bases are inside
- Purple: less coiled (proteins)
- Yellow: attached to ribosome (tRNA)
RNA Characteristics
● RNA can exist in different forms of stability
● Unstable RNA: not a bad thing
○ Its instability plays a role in
achieving different shapes to
accommodate diff. functions
○ Instability DOES NOT refer to
malleability
○ Instability = more likely to break
● Why?
○ RNA is only an ​intermediate
information storage
○ Contributes to its recyclability
○ If too stable, transcript would exist at
all times → proteins produced 24/7
○ This contradicts the ​controlled
expression of proteins
○ Cell does not need to constantly
make something that is not required
at all times → expends too much
energy (wasted)
Differences with DNA
1. Deoxyribose vs. Ribose
- Ribose has an extra OH in the 2’
position
- Makes it ​easier to hydrolyze
- Effect: lower stability
- Counteraction: RNA looks for
binding partners (self, tRNA, rRNA,
proteins)
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- Example: some RNA bind together
to increase their stability such as
tRNA
2. Thymine vs. Uracil
- Uracil is easier to make than
Thymine
- Do not need as extra methyl group
- *Note: some bases in DNA are
converted to Uracil via methylation
- Mutation
- Serves as a marker for
- mistakes in DNA processing
- DNA deletes segment and
recopies the other strand
Miscellaneous questions:
● Why does dsRNA serve as a signal for viral
infection?
● Dicer selection?
● Why Uracil?
Viruses
- Viral RNA create a dsRNA to make itself
more stable
- Dicer selection is a mechanism which
detects double stranded RNA and cleaves it
into smaller RNA strands