Central Dogma and Beyond: Understanding Molecular Diversity from Genomes to Phenotypes

Section 1-1 National Tsing Hua University
CHEM5980
The central Dogma of Molecular Biology
• Information flows from DNA to

RNA then to protein.
• Enzymes (protein) catalyze
reactions in organism that may
involve other biomolecules such
as carbohydrate, polyketide and
terpenes.
CHEM5980
Genes
• Gene is made up with of a promoter and a transcribed sequence
• Transcription factor binds to the promoter of a specific gene to locate the region for
transcription in the genome. This process can activate or repress transcription.
• Therefore gene expression is controlled by the promoter and transcription factor
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Genes
• Related genes are organized into cluster called operon.
• Entire operon is controlled as a group by transcription factor.
• mccA from E.coli is the smallest known gene that encode for heptapeptide microcin A,
which is a toxins precursor produced by bacteria to inhibit the growth of similar or
closely related bacterial strain.
• McC operon includes mccA through mccE to synthesis microcin C7.
• mccF encodes for protein transporter to expel microcin C7 to avoid its toxicity.
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Genomes
• Genome is the collection of all DNA in an organism.
• Whole genome sequencing determines the complete DNA sequence of an organism's
genome.
• Mycoplasma genitalium has smallest genome for a free-living organism at 521 genes;
Human genome contains 20,000 to 25,000 genes.
Species and Common Name Estimated Total Estimated of

Size of Genome Protein-Encoding
(bp)* Genes*
Saccharomyces cerevisiae 12 million 6,000
(unicellular budding yeast)
Trichomonas vaginalis 160 million 60,000
Caenorhabditis elegans 95.5 million 18,000
(nematode)
Drosophila melanogaster (fruit fly) 170 million 14,000
Oryza sativa (rice) 470 million 51,000
Gallus gallus (chicken) 1 billion 20,000-23,000
Canis familiaris (domestic dog) 2.4 billion 19,000
human chimpanzee
Mus musculus (laboratory mouse) 2.5 billion 30,000
Sequence 98.5-99.4% identical
Homo sapiens (human) 2.9 billion 20,000-25,000
Adapted from Nat Edu 2008, 1, 96

CHEM5980
Genomes
• Major accomplishment regarding human genome:
• Sequence the entire genome would help in finding all the genes.
• A organism can be synthesized through the chemical genome synthesis.
• Protein sequences in an organism can be predict from genome, but not the
function.
2001: Human genome sequenced 2010: The first synthetic life form
CHEM5980
Genomes and cell functions
• E. coli is the best understood organism species,

yet our current knowledge still limited.
• Gram negative, commonly found in the lower
intestine.
• Some laboratory strain of E. coli such as K12 are
harmless, while other strains may lead to
infection or food poisoning.
• Rod like shape with diameter slightly more than
1 micron
• 2 layers of membranes with periplasmic space in
between
• Rotating flagella propel E. coli movement
• Contains one copy of genome DNA and several
copies of plasmid, smaller cyclic DNA can be
traded among bacteria.
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Genomes and cell functions

• Human cell are 1000 times larger than E. coli and contain 1000 times more DNA.
• Human DNA are enclose in nucleus by nuclear envelope
• Human cytoplasm contains organelles such as mitochondria, which contains their
own DNA in cyclic form.
• Interestingly, human genome encodes only 5 times more protein than E. coli, the
complexity of multi-cellular organism arise from molecular diversity.
CHEM5980
Cell type cannot be judge by its genomes

• Each human is made up with around 220 types of cells, all the somatic cell with
only few exception have identical genetic identity.
• How the various cell types arise from the same genome during embryonic cell
division belong to the field of developmental biology.
• The differences among various cell types are not due to their genome but the
expression of gene: which turns on or off.
Nerve cell Muscle cell Macrophage
Goblet cell Photoreceptor cell Fat cell

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Phenotype and genotype

• Each gene or combination of genes are called
genotype, while the observable set of
characteristics are called phenotype.
• As an example, antibiotic erythromycin resistance
is phenotype of bacteria, but many genetic Erythromycin
variation can cause phenotype.
mef+ genotype produce special pump to remove
erythromycin.
ermA+ and ermC+ genotype produce enzyme
that modify ribosome to prevent erythromycin
binding.
Single mutation in ribosomal RNA (A2058G) also
make bacteria resistant.
• Current knowledge do not allow to us to predict
the molecular mechanism underneath the
observed phenotypes. There is still a large gap
between DNA sequence and the outcome cellular
properties/functions.
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Beyond genomes: Transcriptome

• Transcriptome is the collection of all RNA transcripts in a cell.
• Transcriptome represents the readout of active gene in a given cell and therefore
depends on tissue type and other variables such as nutrients, extracellular
signaling and temperature.
• Comparing the transcriptome between different cell provide information of which
and the extent of specific gene activation.
• Transcriptome can traditionally read by commercial DNA biochip or newer RNA
sequencing
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Diversity beyond genomes: RNA splicing
• In bacteria, mRNA transcribe are immediately translated into proteins, whereas in

human cells RNA undergo modification called splicing.
• RNA splicing removes a segment of RNA from pre RNA molecule.
• This process cannot be predicted just from the gene sequence, therefore the
protein sequence cannot be predicted solely on DNA gene sequence.
• The splicing process also depend on the environmental conditions. Certain genes
can be spliced into different types on RNAs depending on the other factor.
• The uncertainty in splicing add diversity to a seem straightforward central dogma
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Diversity beyond genomes: Post-translational modifications

• Proteome is the collection of protein
produced in a cell during the next
stage of genetic information flow:
translation
• After ribosomal translation, the
nascent protein can undergo chemical
modifications such as trimming,
phosphorylation, glycosylation…..etc
to further introduce diversity
• In one interesting example,
Corynebacterium diphtheriae product
toxin to modify host elongation factor
EF2, which involves in protein
synthesis.
Addition of a diphthamide moiety of
EF2 shuts down host protein synthesis.
CHEM5980
Diversity from biooligomers without template
• The three other types of biooligomers:

polyketide, oligosaccharides and
terpenes are not encoded or
template-directed
• Use oligosaccharides as example, they
forms branched linkages and the
carbohydrate sequence do not
correlate with linear DNA sequence
• The synthesis different linkages of a
complex oligosaccharide would
require different enzymes encoded by
different genes. The extent of
activation of such gene would also
affect the composition of
oligosaccharide and introduce even
more diversity.
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Combinatorial biosynthesis in the immune system

• The immune system use combinatorial
strategy to create antibodies
• 40 variable (V) modules, 25 diversity (D)
modules and 6 joining (J) modules are
the pool for making antibody variable
region.
• 2 million ways available for combined
heavy and light chains together
• During B-cell selection and maturation,
more detailed arrangement can further
generate antibodies that target specific
antigens
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Diversity from combinatorial assembly
• Linear biooligomers such as DNA, RNA and protein have limited building
blocks and these blocks can be assembled interchangeably. These
characteristics fulfill combinatorial properties.
• For linear oligomers, changing monomer building blocks quickly increase

diversity in a exponential manner. The generated diversity can be estimated
in following equation:
# of possible oligomers = ( # of types of subunits ) length of oligomer
• Such combinatorial principal can be exploit to create a library of DNA

molecules. With the biological machinery, RNA and protein library can be
created accordingly.
Extras National Tsing Hua University
CHEM3100
Complexity of carbohydrate
• Variables in glycosidic linkage includes: monosaccharide types
(aldose or ketose, ring size), which OH involved (C-2’, 3’, 4’ or 6’ of
the glycosyl acceptor), anomeric configuration (α, β)…..
• Branched sugar chains further contribute to complexity
OH
OH
O OH
HO
OH O O
HO OH
OH
O R2 O R4 Possible derivatives
H
H2N N OH
N N
R1
H
O R3
H
O 160,000
Tetrapeptides
O O
84,000,000,000
O O
Tetrasaccharides (branch not estimated)

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Modular architecture for chemical libraries

• When a connecting chemistry has
been established to synthesize
oligomer molecules, building blocks
equipped with the same chemical
handle can be used interchangeably.
This concept of modular synthesis
benefits creation of libraries.
• Using unnatural amino acids as

building blocks, peptoid library can
be created to screen against
interested targets.
• Solid-phase reaction and robotic

synthesis are extremely useful in
making combinatorial libraries due
to handling of large numbers of
compounds. Efficient chemical
reactions are essential for this
strategy.
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Combinatorial chemistry
• How to produce large number of related compounds with relative simple
synthesis strategy?
• The synthesis step must be quick and efficient (high yield)
• Testing compounds must be fast and easy
# of Peptide (based on 20 # of peptides

residues natural amino acids)
2 NH2X1X2COOH 400
3 NH2X1X2X3COOH 8,000
4 NH2X1X2X3X4COOH 160,000
5 NH2X1X2X3X4X5COOH 3,200,000
6 NH2X1X2X3X4X5X6COOH 64,000,000
7 NH2X1X2X3X4X5X6X7COOH 1,280,000,000
8 NH2X1X2X3X4X5X6X7X8COOH 25,600,000,000
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Split-mix synthesis
• Solid support is usually used to aid

purification. Excess reactant can be Split
removed by filtration. React A1 A2 A3
• However even when solid support
used, need as many reaction vessel
A2 A2 A3
as the number of library? A1 A3
A1 A2
A1
• Split-mix method mixes the beads A3
from separated reaction and
redistribute them to different
reactions. A3 A3
Mix A1
• It is possible to screen the compounds, A2 A1
but then how to find out what is the A3
A2 A1
A2
structure of hit?
Split
React B1 B2 B3
A3-B2 A3-B3
A1-B1 A3-B1 A2-B2
A2-B1 A1-B3 A2-B3
A1-B2
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Encoded tag for identification in combinatorial synthesis
PNAS 1997, 94, 2805

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Tools for studying chemical biology: Chromophore

• Most biological molecules are colorless therefore
difficult to quantify or visualize
• Chromophore absorb light at certain wavelength for
quantitative concentration analysis.
• Fluorophore emit light when returns to ground state
and is suitable as marker for quantitative assays and
probes to label specific biomolecules
CHEM5980
Parallel assays and High-throughput screening

• Miniature scale and large number of experiment simultaneously to save reagents and
time--Parallel assay in small scale are required
• Standardized tool invented: microtiter plate, plate reader and multichannel pipette
• Robotic operation to handle large number of samples
• High throughput screening (HTS) processes 100,000 assays per day
Traditional reaction Microarray

Microtiter plate
25mm
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Microbiological screening and selection

• Although high-throughput screen provide fast assay, it is not practical, in terms of
resource and time, when the sample number is just too large.
• Bacteria grow in very large number in limited space and they can replicate to amplify
the selected individual cell.
• Each bacterium can be treated as an individual reaction vessel to perform a massive
screening by its survival or its appearance by phenotype.
• As an example of auxotrophic screening, when an essential protein is deleted in E. coli
and diverse library of millions of variants that expressing different proteins plated in
medium, some bacteria benefit from test protein can grow. These protein can be
identified.
• Weakness to auxotrophic screening
is that natural mutation offers
many unexpected mechanism for
survival.
• Weakness for phenotypic screening
is all strain survives and will take a
lot space for those bacterial
colonies.
CHEM5980
Viruses as tool for gene delivery

• Viruses are organisms that hijack cell machinery to replicate
• Evolution has allow viruses to develop efficient strategy to deliver viral gene
into host cell and for viral enzymes to outperform host enzymes.
• These unique features of viruses were exploited by scientists to manipulate
cell machinery in an efficient manner.
• Virus are also considered as delivery tool for gene therapy.
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Viruses delivery for gene therapy
http://stemcells.nih.gov/StaticResources/info/scireport/images/4_3.jpg
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Viruses as tool for protein screening

• Bacteriophage, virus that infect bacteria, are very useful tool as they make
combinatorial library easily.
• They display corresponding library protein on their surface and can be used for
direct screen against chemical modified surface.
• Selected phage can be amplified easily and be sequenced to characterize the
protein involved in the interaction.
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DNA and RNA aptamer libraries for in vitro screening

• Nucleic acids store genetic information and have great diversity. Advances in chemical
or biological methods allows manipulation of these molecules.
• Can DNA and RNA play other roles based on these advantages?
• Specific nucleic acids can be selected by the random change in sequence to bind small
molecules. These group of biopolymers are called aptamers.
• Systematic evolution of ligands by exponential enrichment (SELEX) is one
of the technology to find such aptamers against specific small molecules.
Nat Protocol 2006, 1, 2204

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Small molecule inhibitors

• Small molecules play important roles in identification the functions of specific
proteins and to manipulate the biological systems.
• The advantage of small molecules is their membrane permeability. This property
allows perturbation of biological processes within a cell. These tools can be
exploited to switch on/off certain pathway, identify the function of certain protein
and more.
• Selectivity is the essential
property for these small
molecule tools. A selective
effector allow control of a
specific pathway without
altering others. It is also
necessary to avoid
triggering unwanted
protein, during the
investigation of protein
functions.
CHEM5980
Small molecule inhibitors
Sphingosine 1-phosphate
natural ligand SEW2871 agonist
W146 antagonist
Nat Chem Biol 2006, 2, 434

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Prevention of gene expression

• To study the function of specific gene (or the expressed protein), one straightforward
strategy is to remove this protein and observe the change.
• The traditional method is to knockout the target gene by DNA recombination. This
time and labor consuming method provide stable gene deleted cell line/organism.
• More recently, RNA interference was discovery and exploited to transiently stop
synthesis of specific protein in a very convenient manner.
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Monoclonal antibody
• Antibodies are known for their specific and strong bonding to specific molecules
• When B cell encounter foreign antigen, they respond to different epitopes, as the
result, the generated antibodies are specific for different epitopes
• To use antibody as tool, they must be monoclonal that behave in the same way
How to improve from polyclonal to monoclonal?
Separate B cells one by one then fuse to immortal hybridomas and produce antibody
B cell
B cell receptor
antigen
Monoclonal epitope Polyclonal
antibody antibody
Antibodies from HIV infected patients
Neutralization potency
(µg/ml)
Protocol G:
To generate broadly neutralizing Donor 17
monoclonal antibodies (mAbs)
from volunteers who are HIV infected and
have broadly cross-reactive serum
neutralizing activity. Donor 36
• Sera from 1,800 HIV infected donor

screened for neutralization breadth
Donor 39
and potency (162 strains of HIV)
• mAbs isolated from top 4 elite donors

Donor 84
• Many of the PGT antibodies are 10-
fold more potent than recent isolated
PG9, PG16 and VRC01 Existing
broad
neutralizing
mAbs
Nature 2011, 477, 466-470.

With
Characterization of PGT antibodies
• Competition with existing mAb help identify the location of epitopes

• Gp120 mutant (Alanine scan) pin-points PGT antibodies binding sites
25000
Man4NH2
20000
Man4
15000
Man8NH2
Intensity
Man8
10000
Man9NH2
5000 Man9
0
121 122 123 125 126 127 128 130 131 135 136 137 141 142 143 144 145 2G12
Man9GlcN
Ac2Man9GlcNAc2
PGT antibody #
• PGT 125-128 and 130 initiate strong binding to branched oligomannose

• Unlike 2G12 the new PGT antibodies do not bind Man4
With
Characterization of PGT antibodies
Inhibition assay design Man4 dendron inhibition

Fluorescent
2nd Ab
2G12
gp120
Glass slide Man9 dendron inhibition
• Man9, but not Man4, dendron inhibits

gp120 binding to PGT125-128 and PGT130.
Indicate Man8/9 fragment are primary
epitope.
• Multiple mutation experiments indicate
that glycan at N332 and N302 of gp120
are important for neutralization
With Nature 2011, 477, 466-470.

Structural studies of PGT128 Fab-gp120 outer domain complex
• Resolution at 3.25Å
• Binding contributed from 3 subsites:
N332 glycan, N301 glycan and
C-terminal of V3
• N332 Man8/9GlcNAc2 binds PGT128 in a similar
manner as synthetic Man9.
Buried surface area: 449Å2
• N301 glycan bind PGT128 through its

Man5GlcNAc2 core.
328Å2
• C-terminus of V3 bind contact with PGT128

are primarily through backbone interaction
that are tolerant of side chain variations.
305Å2
Science 2011, 334, 1097-1103

With
CHEM5980
Human cell lines

• Some common mammalian cell lines are derived from cancer cells. Although
these immortal cell may not be exactly the same as the healthy tissue, they are
convenient system as human organ mimic.
• When cellular experiments are conducted, it is very important to point out what
cell line was used.
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Human stem cells

• Many cells called stem cells have capacity to regenerate various of tissue type.
• Advances in stem cell
research have demonstrated
their potential in
regenerative medicine by
replacing damaged tissues.
• The most potent stem cells
come from embryo. To avoid
moral controversy, http://www.stemcelltreatments.in/contact_us
researchers looked for
method to produce potent
stem cells without using
embryo.
• Induced pluripotent stem
(iPS) cells have been
developed by using adult
cells to produce embryonic -
like stem cells.
CHEM5980
Source of stem cells
http://www.frcblog.com/wp-content/uploads/2010/07/stemcells-fert-clone-iPS-ASC2.jpg
CHEM5980
Source of stem cells
J Am Chem Soc 2004, 126, 410

http://www.sigmaaldrich.com
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iPS application
Nature 2008, 453, 322

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Human stem cells
• Retrovirus exploited as vector for Oct3/4,

Sox2, c-Myc, and Klf4 that encode
transcription factors.
Cell 2006, 126, 663

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Human stem cells
• In the case that Oct3/4, Sox2, c-Myc, and

Klf4 gene may cause problems, the
expressed protein may avoid them.
• Fusion the transcription factors with cell
penetrating peptide help the delivery of the
protein.
Cell Stem Cell 2009, 4, 381

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Model organisms for research

• The research on human related topics are important, however the experiment
on human are always limited due to ethical and practical reasons.
• Models from other species often are used to simplify the experiments
• These model organisms span from prokaryotes to eukaryotes, from single cell to
multiple cells, from insects to mammals.
• The advantage using these models include the cost and time. Such organisms
are easy to grow/breed and their generation time is short to provide quick
results.
CHEM5980
Model organisms for research
• Single-cell organisms including bacteria and yeast provide

excellent tool for research at the molecular level such as
DNA production and protein expression.
• Fruit fly and round worm have 14,000 and 20,000 genes
respectively that is close to human. These are good model
for developmental and behavior studies.
• Effects of alcohol on fruit flies are similar to human response.
• Mice are essential for studying behavior, metabolism and
immunity. Drugs and new treatment are usually first tested
on mice before moving to higher animal models.
CHEM5980
Animal experiments

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Section 1-1 National Tsing Hua University

The central Dogma of Molecular Biology

• Information flows from DNA to

Species and Common Name Estimated Total Estimated of

Adapted from Nat Edu 2008, 1, 96

Genomes and cell functions

• E. coli is the best understood organism species,

Genomes and cell functions

Cell type cannot be judge by its genomes

Nerve cell Muscle cell Macrophage

Goblet cell Photoreceptor cell Fat cell

Phenotype and genotype

Beyond genomes: Transcriptome

Diversity beyond genomes: RNA splicing

• In bacteria, mRNA transcribe are immediately translated into proteins, whereas in

Diversity beyond genomes: Post-translational modifications

Diversity from biooligomers without template

• The three other types of biooligomers:

Combinatorial biosynthesis in the immune system

Diversity from combinatorial assembly

• For linear oligomers, changing monomer building blocks quickly increase

# of possible oligomers = ( # of types of subunits ) length of oligomer

• Such combinatorial principal can be exploit to create a library of DNA

Tetrasaccharides (branch not estimated)

Modular architecture for chemical libraries

• Using unnatural amino acids as

• Solid-phase reaction and robotic

# of Peptide (based on 20 # of peptides

• Solid support is usually used to aid

Encoded tag for identification in combinatorial synthesis

PNAS 1997, 94, 2805

Tools for studying chemical biology: Chromophore

Parallel assays and High-throughput screening

Traditional reaction Microarray

Microbiological screening and selection

Viruses as tool for gene delivery

Viruses delivery for gene therapy

Viruses as tool for protein screening

DNA and RNA aptamer libraries for in vitro screening

Nat Protocol 2006, 1, 2204

Small molecule inhibitors

Small molecule inhibitors

Nat Chem Biol 2006, 2, 434

Prevention of gene expression

• Sera from 1,800 HIV infected donor

• mAbs isolated from top 4 elite donors

Nature 2011, 477, 466-470.

• Competition with existing mAb help identify the location of epitopes

• PGT 125-128 and 130 initiate strong binding to branched oligomannose

Inhibition assay design Man4 dendron inhibition

Glass slide Man9 dendron inhibition

• Man9, but not Man4, dendron inhibits

With Nature 2011, 477, 466-470.

• N301 glycan bind PGT128 through its

• C-terminus of V3 bind contact with PGT128

Science 2011, 334, 1097-1103

Human cell lines

Human stem cells

Source of stem cells

Source of stem cells

J Am Chem Soc 2004, 126, 410

Nature 2008, 453, 322

Human stem cells