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Chapter 5: DNA
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The structure of DNA allows efficient storage
of genetic information.
Genetic information in DNA can be accurately
copied and can be translated to make the
proteins needed by the cell.
The structure of DNA is ideally suited to its
function
Information stored as a code in DNA is copied
onto mRNA
Information transferred from DNA to mRNA is
translated into an amino acid sequence.
I. Is DNA the genetic material?
For many years it was thought that proteins and not
DNA contained the genetic material.
A. Experiment by Alfred Hershey and Martha Chase in
1952 confirmed DNA as genetic material
1. Used radioisotopes: radioactive forms of
elements that decay at a predictable rate and
allows for detection of the element
2. Grew bacteriophage viruses in two different
cultures
a) One with radioactive phosphorus 32P:
phosphorus is in DNA
b) One with radioactive sulfur 35S: sulfur in
protein coat and not DNA
3. When the virus infected the bacteria, the phosphorus
could be detected inside the bacterium and not sulfur
4. Hersey and Chase concluded that DNA, not protein,
was the genetic material
5. Research involving genetics centered on DNA
II. Structure of DNA
A. Types of nucleic acids: ATP, DNA, and RNA
1. ATP: energy storage
2. DNA and RNA: stores genetic information
B. Nucleotides: monomer that makes up the DNA and
RNA polymers and consists of three parts
1. Phosphate group: circle in diagrams
2. 5-Carbon monosaccharide (sugar-pentose):
pentagon in diagrams
a) DNA: deoxyribose sugar
b) RNA: ribose sugar
3. Nitrogenous bases: rectangle in diagrams
a) A-adenine
b) C-cytosine
c) G- guanine
d) T-thymine (DNA)
e) U-uracil (RNA)
C. Monomers into polymers
1. Nucleotides attach to each other by
condensation reactions to form covalent bonds in
order to produce a long chain
a) Phosphodiester bond: forms between the
hydroxyl group of the 3’ C of deoxyribose and
phosphate group attached to the 5’carbon of
deoxyribose
2. Chain alternates the pentose sugars and
phosphate to form the backbone
3. Nitrogenous bases extending outward
D. Single or double strand
1. RNA is a single strand of
nucleotides
2. DNA is a double strand of nucleotides
(forms a ladder structure)
a) Nitrogen bases are connected by
hydrogen bonds to form the rungs of
the ladder
3. Complementary base pairing
a) A and T pair with double hydrogen bond
b) G and C pair with a triple hydrogen bond
E. Model of the DNA strand
1. Antiparallel strands
a) One strand
i) 5-carbon (5-prime or 5’) unattached
at the top of the strand
ii) 3-carbon (3-prime or 3’) unattached
at the bottom of the strand
b) Other strand: 3’ at top and 5’ at bottom
2. Condensation reaction: water molecule is
released when there is a reaction between the
phosphate group on the 5’ carbon and the
hydroxyl (OH) group on the 3’ carbon
3. Each time a nucleotide is added, it is
attached to the 3’carbon end using the
phosphodiester bond
4. Hydrogen bonds link the nitrogenous
bases together to hold the two sugarphosphate backbones together
a) Purines: adenine and guanine have a
double ring structure
b) Pyrimidines: cytosine and thymine have
a single ring structure
Purines
Adenine
Guanine
Phosphate
group
Pyrimidines
Cytosine
Thymine
Deoxyribose
c) Complementary base pairing: occurs because
of the specific distance that exists between the two
sugar-phosphate chains
i) Adenine always pairs with thymine
using two hydrogen bonds
ii) Guanine always pairs with cytosine
using three hydrogen bonds DNA
Draw a diagram of a DNA ladder in which the
nitrogenous base sequence of one strand is
C,T,G,G,A,T,C,A,G, T.
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The first cytosine in the sequence should be at the 5’
top of the ladder
Draw using the diagram shapes: circle, pentagon,
and rectangle
Indicate the 5’ and 3’ ends and show that the strands
are antiparallel
Use the correct number of hydrogen bonds between
the complementary nitrogen bases
Color code your diagram and give a key at the
bottom of the page
5. Double helix shape formed by electrical charges
causing the DNA to twist
6. James Watson (American) and Francis Crick
(British) proposed the double helix shape in 1953
by making a physical model based on data from
many sources
7. Other scientists’ contributions
a) Erwin Chargaff (Austrian): determined A
and T were always equal
b) Rosalind Franklin (British) and Maurice
Wilkins (New Zealand): calculated distance
between various molecules in DNA by X-ray
crystallography
Side note on the competition between three groups of
scientists working on the structure of DNA
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Openly competed to determine the structure of
DNA
Watson and Crick from Cambridge
Maurice Wilkins and Rosalind Franklin from Kings
College in London
Linus Pauling’s team at Caltech in the US
Watson, Crick, and Wilkins shared the Nobel Prize
Within research groups, collaboration is important,
but group competition often restricts open
communication (could limit or increase discoveries)
III. DNA packaging
A. Histone proteins: Several kinds of circular histones that
help in DNA packaging
1. Packaging is essential for the DNA to fit inside the nucleus
because a single human molecule of DNA can be 4 cm long
2. Nucleosome: consists of 2 molecules of each of four
different histones (total of 8) and DNA wraps twice around
each of the eight histones
a) DNA is negatively charged and histones are positively
charged, so they are attracted to each other
b) Between the nucleosomes is a single string
of DNA
c) Fifth type of histone is attached to the
linking string of DNA near each nucleosome
and leads to further wrapping of the DNA
molecule
Packing DNA
Histones
iPad Activity
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Go to www.rcsb.org/pdb/home/home.do
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At search box: nucleosome
Scroll down to Molecule of the Month and
select nucleosome
 Under Exploring structure click 1aoi in the
text
 On the right, click on 3D view
 Display mode (toggle between them)
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IV. DNA Replication-Overview
A. DNA replication: cells must double their DNA
content in order to prepare for cell division
1. Makes an exact copy of the DNA
2. Two types of molecules in nucleus that are
important for replication
a) Enzymes: helicases and DNA
polymerases
b) Free nucleotides: floating freely in
nucleoplasm
3. Watson and Crick realized that the A-T and C-G
base pairing provided a way for DNA to be copied
a) Semi-conservative model of DNA
replication: single strand of DNA could serve
as a template for a copy of the DNA
b) Meselson and Stahl conducted experiments
in the 1950’s that confirmed the DNA semiconservative model (see worksheet)
B. Steps of replication
1. Separation of the double helix into two single strands
a) Helicase: enzyme that initiates the separation
by starting at a point in or at the end of the DNA
i) moves one complementary base pair at
a time
ii) breaks the hydrogen bond between the
bases
b) Like a zipper: helicase is the slide mechanism
and the two sides of the DNA is like the two opened
sides of a zipper
2. Nitrogenous bases on each strand is now unpaired and
can be used as a template to create two double-stranded
DNA molecules identical to the original
3. DNA polymerase: enzyme that takes a free floating
nucleotide and joins it to unpaired nucleotide by forming
a covalent bond
4. Process continues to add nucleotides on both of the
original DNA strands
a) One strand replicates in the same direction as
the helicase is moving
b) Other strand replicates in the opposite direction
5. Two identical DNA strands are produced
a) Every DNA molecule consists of a strand
that is “old” paired with a strand that is “new”
b) Semi-conservative process: half of a preexisting DNA molecule is always conserved or
saved
DNA replication
V. DNA Replication: Detailed steps
A. Difference between prokaryotes and eukaryotes
Prokaryotes
Eukaryotes
Circular DNA
Linear DNA
No histones
Histones
Single origin of replication
Thousands of origins of
replication
Shorter/smaller
Longer/larger
B. Separating the two strands
1. Origin of replication: special sites where
DNA replication starts, appears as a bubble
where the two strands are separated
2. Helicase: enzyme that unzips the strands by
breaking the hydrogen bonds
3. Replication fork: where the double stranded
DNA opens to provide the template by the parent
strands
C. Elongation of a new DNA strand (Continuous
Synthesis)
1. Primer is produced by primase (enzyme) at the
replication fork
a) Primer: short sequence of RNA (5-10
nucleotides)
b) Primase: enzyme that joins the RNA
nucleotides that match the exposed DNA bases
2. DNA polymerase III: adds nucleotides in a 5’ to 3’
direction to produce the growing DNA strand
3. DNA polymerase I: removes RNA primer from the 5’
end and replaces it with the DNA nucleotides
D. Deoxynucleoside triphosphate (dNTP): actual
nucleotide that is added to elongating DNA strand
1. Contains deoxyribose, nitrogenous bases
(A,T,C or G) and three phosphate groups
2. As the molecules are added, two phosphates
are lost which provides energy needed for the
chemical bonding of the nucleotides
E. Antiparallel strand formation for the 3’ to 5’
template
1. Leading strand: 5’ to 3’ strand is produced
continuously and relatively fast
2. Lagging strand: 3’ to 5’ strand is produced in
fragments and more slowly
3. DNA polymerase III can only work in the 5’
to 3’ direction
F. Steps for the elongation of the lagging strand
(Discontinuous synthesis)
1. Primer is added to 3’ to 5’ strand using primase
2. DNA polymerase III adds several nucleotides
to produce Okazaki fragments
3. DNA polymerase I removes primer and replaces
it with DNA nucleotides
4. DNA ligase: enzyme that joins the Okazaki
fragments by attaching the sugar-phosphate
backbones of the lagging strand fragments to form a
single DNA strand
Animation
Roles of replication enzymes in bacteria (E. coli)
Protein/enzyme
Role
Helicase
Unwinds the double helix at replication forks
Primase
Synthesizes RNA primer
DNA Polymerase III
Synthesizes the new strand by adding nucleotides onto
the primer (5’to3’)
DNA Polymerase I
Removes primer and replaces it with DNA
DNA ligase
Joins ends of DNA segments and Okazaki fragments
Replication in eukaryotes and prokaryotes is almost identical
• Eukaryotes: DNA gyrase: stabilizes the DNA helix when
helicase unzips
G. Speed and accuracy of replication
1. Speed: 4000 nucleotides are replicated per second
2. Bacteria can divide in 20 minutes so speed of
replication is essential
3. Eukaryotic cells have a huge number of
nucleotides compared to prokaryotes so multiple
replication origins are needed
4. Replication is accurate: few errors (mutations
occur)
a) repair enzymes are used detect and
correct errors
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Bio Alive website links
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Replication
VI. Human Genome Project
A. Overview
1. Human genome: the complete nucleotide
sequence in humans
2. 3.2 billion base pairs in the 23 human
chromosomes
3. Now scientists need to understand what the
DNA sequences encode
4. Bioinformatics: used computers to compare
different DNA sequences
5. Will help to diagnose, treat, and prevent genetic
disorders, cancer and infectious diseases in the
future.
B. 1970’s Frederick Sanger developed the first sequencing procedure
1. Polymerase chain reaction (PCR): uses fragments of DNA and
produces a large number of copies and then denatured (separated in single
strands) by heating to 92 °-94° C
a) Can be studied and analyzed and often used in forensics
when a limited amount of DNA has been recovered
b) Thermus aquaticus (Taq): bacteria that produces an
enzyme that is stable a high temperatures and is not
denatured at high temperatures needed for PCR
i) Found in the hot springs at Yellowstone National Park
ii) Taq polymerase: DNA polymerase that has greatly
increased the number of discoveries
Mushroom Pool at
Yellowstone National Park
Thermus aquaticus
2. Sequencing Steps
a) Single stranded fragments are placed in 4 test
tubes with primers, DNA polymerase, and
nucleotides
b) Each tube has special nucleotide called
dideoxynucleotide, derived from dideoxynucleic
acid (ddNTP): after being added by DNA
polymerase, it prevents any further nucleotide
addition to the chain (4 types: A,T,C,G)
c) Synthesis of each new DNA strand begins at 3’
end of primer and continues until dideoxynucleotide
is added (allows for various lengths)
d) DNA for each tube is placed in gel
electrophoresis: bands produced may be used to
determine the exact sequence of fragment of
DNA
C. Newer methods of DNA sequencing are faster
and cheaper today
1. Dideoxyribonucleic acid and ddNTPs are still
used but now labelled with fluorescent markers
for easy recognition and quicker sequencing
2. New methods allowed faster results with the
Human Genome Project
Genome project
VII. Central dogma of molecular biology and RNA
A. Francis Crick in 1956 proposed the central
dogma (Flow of genetic information)
1. DNA RNA Proteins
a) Transcription: DNA makes RNA
b) Translation: RNA makes the protein
c) Protein gives the characteristic-does the
work of the cell
Genome video
VIII. RNA
A. RNA Function
1. RNA carries the genetic information
from the nucleus (DNA) to the cytoplasm
2. The proteins are then made in the
cytoplasm from the instructions in the RNA
B. Types of RNA: each has a different function
1. Messenger RNA (mRNA): carries genetic
information from the DNA in the nucleus to the
cytoplasm
2. Transfer RNA (tRNA): strand folded into a
hairpin shape that binds to specific amino acids
3. Ribosomal RNA (rRNA): globular form that
combines with proteins to make the ribosomes where
proteins will be made
RNA types
IX. Transcription: making RNA from DNA in the
nucleus
A. Steps
1. RNA polymerase: enzyme that unzips
DNA (small portion-gene) like helicase and
adds complementary RNA nucleotides
(nucleoside triphosphates (NTPs)) using
the DNA as the template
2. Only one strand is used as the template
a) Sense strand: DNA strand that carries the
genetic code (Same sequence as the newly
transcribed RNA except for thymine and uracil)
b) Antisense strand: template strand that is
copied during transcription
3. 5’ ends of free RNA nucleotides are added
to the 3’ end of the RNA molecule being made
4. Promoter: area of the DNA where the
RNA polymerase attaches
5. Continues to add RNA nucleotides as the
transcription bubble moves
6. Terminator: sequence of nucleotides that
causes the RNA polymerase to detach from the
DNA
7. Transcription stops and new mRNA is
detached from the DNA
Note: In eukaryotes, transcription continues beyond
the terminator for a significant number of
nucleotides before it is released.
B. Important facts of transcription
1. Promoter transcription unit terminator
2. Only one strand of DNA is copied
3. mRNA is single-stranded and shorter than DNA
since it is copying only one gene to make one protein
4. RNA has ribose and uracil
5. DNA has deoxyribose and thymine
Transcription
Transcription clip (Castle)
C. Genetic code:
DNA triplet transcription mRNA codon
1. Message written in the mRNA determines the
order of the amino acids that will make up the protein
2. Codon : three bases of mRNA that
determines the type of amino acid
3. Anticodon: three bases on the tRNA that
complementary pair up with the codon of mRNA
a) tRNA picks up the specific amino acid
b) 20 amino acids
The Genetic Code
D. Post-transcription modification of mRNA
1. Introns: stretches of non-coding DNA in
eukaryotes
2. Exons: coding sequences of mRNA
3. Splicing: removing the introns to make a
functional mRNA strand
4. Spliceosomes: small nuclear RNAs (snRNAs) that
remove the introns
a) The exons may be rearranged during splicing
resulting in different possible proteins
b) Different sections of a gene act as introns at
different times which increases the number of
proteins that can be made by one gene
5. The 5’ end of mature mRNA has a cap of modified
guanine nucleotide with three phosphates
6. The 3’ end has a poly-A tail: 50-250 adenine
nucleotides
8. The modified two ends protect the mature mRNA
from degradation in the cytoplasm and enhances the
translation process at the ribosome
Splicing
E. Gene expression
1. Methylation: adding methyl group (CH3)
a) Inactive DNA is usually highly
methylated and usually not transcribed or
expressed
b) Gene stays methylated through many
cell divisions
c) Methyl group seems to cause a section of
DNA to wrap more tightly around histones
which prevents transcription
d) Methylation may regulate the
expression of either the maternal or
paternal form of the gene
f) Methylation patterns have been
associated with large number of cancers
and the patterns are being used in the
diagnosis and treatment of some cancers
2. Proteins and gene expression
a) Transcription factors: proteins that
regulate transcription by assisting the binding of
RNA polymerase at the promoter region of the
gene
b) Transcription activators: protein that
causes looping of DNA, which results in
shorter distance between the activator and
promoter
c) Repressor proteins: bind to segments of DNA
called silencers and this prevents transcription of that
segment
Expression clip
3. Environment and gene expression
a) Evidence that the environment can determine
what genes are expressed
b) Study showed that many more respiratory
genes are expressed in people living in urban
areas (pollutants in urban areas stimulate the
problems of asthma because of the expression of
usually non-expressed genes)
4. Epigenetics: study of a set of reversible heritable
changes that occurs without a change in DNA nucleotide
sequence
a) Studies: splicing, methylation, proteins, and
environment Epigenetics
X. Translation
A. Ribosomes: organelle for protein synthesis
1. Consists of large subunit and small
subunit composed of rRNA and proteins
2. Ribosomes are made in the nucleolus of
eukaryotic cells and move through the
membrane pores
3. Translation occurs in the space between the
two subunits
4. Binding sites on the ribosome
Site
Function
A
Holds tRNA carrying the next amino acid to be added
to the polypeptide chain
P
Holds the tRNA carrying the growing polypeptide
chain
E
Site from which tRNA that has lost its amino acid is
discharged
5. Triplet bases of the mRNA codon pair with
complementary bases of triplet anticodon of
tRNA
6. tRNA moves sequentially through the three
binding sites from A, to P, to E site
7. Growing polypeptide chain exits the ribosome
through a tunnel in the large subunit
B. Genetic Code
1. 64 possible codons
a) Three are stop codons: UAA, UAG, UGA
b) AUG: start and for methionine
2. Genetic code is degenerate: for each amino acid,
there may be more than one codon
3. Genetic code is universal: all organisms share the
same code
a) Allows for genetic engineering: exchange of
genes from one species to another
Translation steps: Initiation, elongation,
translocation, and termination
C. Initiation
1. Start codon (AUG) on 5’ end of all mRNA
2. tRNA: 3’ end is free and has sequence CCA
and this is the site of amino acid attachment:
one of the loops has the anticodon that pairs
with the specific mRNA codon
3. 20 different amino acids will bind to the correct
tRNA because of the action of 20 different enzymes
a) Active site of each enzyme allows a fit only
between one specific amino acid and the
specific tRNA
b) Requires energy provided by ATP
c) Activated amino acid and tRNA can deliver
it to a ribosome
4. Activated amino acid, methionine attached to a tRNA
with anticodon UAC, combines with mRNA and a
small ribosomal subunit
5. Small subunit moves down the mRNA unit it
contacts the start codon (AUG)
6. Hydrogen bonds form between the initiator tRNA
and start codon
7. Large ribosomal subunit combines with these parts to
form the translation initiation complex
8. Initiation factors: proteins that join complex and
require energy from GTP (like ATP)
D. Elongation phase
1. Involves tRNAs bringing amino acids to the
mRNA complex in the order specified by the codons
2. Elongation factors: proteins that assist in binding
the tRNAs to the mRNA codons at the A site
(attachment site)
3. Initiator tRNA moves to the P site (parking site)
4. Ribosomes catalyze the formation of peptide
bonds between adjacent amino acids
a) Condensation reaction produces the
peptide bond and water is formed
E. Translocation phase
1. Happens during elongation phase
2. Movement of the tRNA from one site of
mRNA to another
a) tRNA binds to A site and its amino
acid is then added to the growing
polypeptide chain by a peptide bond
b) tRNA moves to P site and transfers its
polypeptide chain to new tRNA which
came into the A site
c) Empty tRNA moves to the E site (exit
site) and it is released
3. Process occurs in the 5’to 3’ direction
4. Ribosomal complex is moving along mRNA
towards the 3’ end
F. Termination phase
1. Begins when one of the three stop codons appears at
the open A site
2. Release factor: protein fills the A site and doesn’t
have an amino acid
a) Release factor catalyzes hydrolysis of the bond
linking the tRNA in the P site with the polypeptide
chain
b) Frees the polypeptide, releasing it from the
ribosome
3. Ribosome then separates form the mRNA
and splits into its two subunits
4. Translation is complete and proteins have
several different destinations
a) Free ribosomes proteins are used
within the cell
b) Ribosomes on the endoplasmic
reticulum are secret from the cell or used
in lysosomes
5. Polysome: string of ribosomes going
through the process of translation on one
mRNA at the same time
Translation
Nucleus
Messenger RNA
Messenger RNA is transcribed in the nucleus.
Phenylalanine
tRNA
mRNA
Transfer RNA
Methionine
The mRNA then enters the cytoplasm and
attaches to a ribosome. Translation begins at
AUG, the start codon. Each transfer RNA has
an anticodon whose bases are complementary
to a codon on the mRNA strand. The ribosome
positions the start codon to attract its
anticodon, which is part of the tRNA that binds
methionine. The ribosome also binds the next
codon and its anticodon.
Ribosome
mRNA
Lysine
Start codon
Translation (continued)
The Polypeptide “Assembly Line”
The ribosome joins the two amino acids—
methionine and phenylalanine—and breaks
the bond between methionine and its tRNA.
The tRNA floats away, allowing the ribosome
to bind to another tRNA. The ribosome moves
along the mRNA, binding new tRNA molecules
and amino acids.
Lysine
Growing polypeptide chain
Ribosome
tRNA
tRNA
mRNA
Completing the Polypeptide
mRNA
Ribosome
Translation direction
The process continues until the ribosome reaches
one of the three stop codons. The result is a
growing polypeptide chain.
Translation (Castle)
Translate and Transcribe
You Tube
XI. Protein functions and structures
A. Protein function is closely tied to its structure
1. Four levels of organization of protein
structure
2. Primary, secondary, tertiary, and
quaternary
B. Primary organization
1. Unique sequence of amino acids attached by
peptide bonds and determined by the nucleotide
base sequence in the DNA
2. Every organism has its own DNA, so every
organism has its own unique proteins
3.Determines the next three levels of
organization: changing one amino acid could
completely alter the structure and function of
the protein
Example: Sickle cell disease Sickle cell
C. Secondary organization
1. Created by formation of hydrogen
bonds between the oxygen from
carboxyl group of one amino
acid and the hydrogen from
the amino group of another
amino acid
2. Two most common shapes are
alpha-helix (α-helix) and
beta-pleated sheet (β-pleated sheet)
D. Tertiary organization
1. Polypeptide chain bends and folds over itself
because of interaction among the R-groups (side
groups) and the peptide backbone
2. 3D conformation
3. Interactions
a) Disulfide bonds: covalent bonds between
sulfur atoms, called bridges because they are
strong bonds
b) Hydrogen bonds between polar side chains
c) Van der Waals interactions between
hydrophobic side chains of amino acids (strong)
d) Ionic bonds between positively and negatively
charged side chains
4. Tertiary structure is important in determining the
specificity of proteins that are enzymes
E. Quaternary organization
1. Involves multiple polypeptide
chains that combine to form a single
structure
2. Not all proteins have quaternary structure
3. All bonds from the first three levels are involved
in this level
4. Conjugated proteins: contain a prosthetic or
non-polypeptide group
a) Hemoglobin (haemoglobin): contains for
polypeptide chains and each contains a nonpolypeptide group called haem (heme)
b) Haem (heme): contains an iron atom that
binds to oxygen
c) Hemoglobin is found in red blood cells and
carries the oxygen
Structure
Structure
F. Fibrous and globular proteins
1. Fibrous proteins: composed of many polypeptide
chains in a long, narrow shape
a) Usually insoluble in water
b) Example: Collagen: structural role in the
connective tissue of humans
c) Example: Actin: component of human
muscles and is involved in contractions
2. Globular proteins: 3D in their shape and mostly
water soluble
a) Example: hemoglobin
b) Example: insulin (regulating blood sugar levels)
Pictures
G. Polar and non-polar amino acids: Group based on their
side chains (R-groups)
1. Non-polar side chains: hydrophobic
2. Polar: hydrophilic properties and found in areas of proteins
that are exposed to water
a) Membrane proteins have polar amino acids toward
the interior and exterior of the membrane and create
hydrophilic channels in proteins through which polar
substances can move
3. Important in determining the specificity of an enzyme
a) Active site on enzyme and specific substrates must
fit together based on shape and polarity properties