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Unit 4: DNA and Protein Synthesis
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The History of DNA
Is DNA or protein the genetic material of life?
• Frederick Griffith (1928) – Identified a “transformation
factor” that can cause new traits to be inherited in
bacteria
– Harmless bacteria became disease-causing once exposed to the
remains of dead disease-causing bacteria
• Hershey and Chase (1952) – studied bacteriophages
(viruses that attack bacteria) and discovered that DNA is
the genetic material of life
– Used radioactive isotopes as tracers to track the movement of
proteins and DNA from bacteriophages into bacteria
How is genetic information
inherited?....DNA
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Genetic information is passed from one cell to the next
by a molecule called DNA. DNA is located inside
chromosomes within the nucleus of a eukaryotic cell.
What does DNA stand for? Deoxyribonucleic Acid.
DNA is a nucleic acid made of two strands of nucleotides
wound together in a spiral called a double helix. The two
strands are antiparallel, which means they run in
opposite directions. One strand runs 5’ to 3’ and the
other runs 3’ to 5’.
Each nucleotide is composed of the following:
1.) a sugar molecule known as
deoxyribose
2.) a phosphate group
3.) one of four different nitrogenous
bases:
adenine (A)
thymine (T)
guanine (G)
cytosine (C)
DNA Structure
• The nitrogen containing bases are the only difference in the four
nucleotides.
• DNA structure is the same in all organisms. The DNA of different organisms
differs ONLY in the sequence of nucleotides. The differences in nucleotide
sequence are responsible for the genetic differences between organisms.
These nucleotide sequences hold the code used to make proteins.
DNA Structure
• Watson and Crick (1953), along with the help of Rosalind Franklin and
Maurice Wilkin, determined the three-dimensional structure of DNA by
building models according to Franklin’s images of DNA taken by X-ray
crystallography.
• They realized that DNA is a double helix that is made up of an alternating
sugar-phosphate backbone on the outside with nitrogen bases on the
inside.
DNA Structure
• Nucleotides always pair in the
same way. They follow Chargaff’s
rule, also known as the base pair
rule, which states….
– A binds with T
– C binds with G
• Because a pyrimidine (single ring)
pairs with a purine (double ring),
the helix has a uniform width.
• The backbone (sugars and
phosphates) is connected by
covalent bonds called
phosphodiester bonds. The
nitrogenous bases are connected
by hydrogen bonds.
View this video on DNA Basics!
DNA Replication
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DNA Replication is the process in which DNA makes an exact copy of itself.
A single strand of DNA serves as a template (pattern) for a new strand.
Replication occurs following the base-pair rules (A = T and C = G).
DNA is replicated during Interphase (S – Synthesis) of the cell cycle.
Each somatic cell (body cell) gets a complete set of identical DNA.
DNA Replication
• DNA Replication is semi-conservative which means that the DNA molecule
uncoils and separates into two strands. Each original strand becomes a
template on which a new strand is constructed, resulting in two DNA
molecules identical to the original DNA molecule.
One strand is part of the
original parent strand and one
strand is a newly formed
strand…hence the name semiconservative (one strand was
conserved).
Step 1: The enzyme helicase unzips DNA in multiple locations, called
origins of replication, within the DNA double helix forming what is
referred to as the replication fork.
nucleotide
The DNA molecule unzips
in both directions.
There are many origins of replication in eukaryotic chromosomes.
Step 2: Nucleotides floating within the nucleus of the cell attach to
complementary nucleotides from the original unzipped strands. The
enzyme DNA polymerase forms the new daughter strand from the 5’
end to the 3’ end of the leading strand. The strand that runs 3’ to 5’ is
called the lagging strand and it is copied in a series of segments called
Okazaki fragments, which are then bound together by DNA ligase.
new strand
nucleotide
DNA polymerase
Step 3:Two new molecules of DNA are formed, each with an original strand
and a newly formed strand. Once again, this is why we say replication is
“semi-conservative.”
original strand
new strand
Two molecules of DNA
The enzyme DNA polymerase can find and remove incorrect nucleotides. DNA
polymerases and DNA ligase can also correct errors to prevent mutations from
occurring.
DNA Replication
A new strand only grows from 5’ to 3’ end.
Nucleotides are added at a rate of approximately 50 per second in mammals
and 500 per second in bacteria.
Protein Synthesis & Gene Expression
DNA  RNA Protein  Traits
– Genetic information flows in one direction from DNA to
RNA to proteins…this is called the Central Dogma. This
flow of genetic information determines what traits you
express. Three important processes occur in cells to allow
this flow of genetic information to occur:
• Step 1 : Replication
replication
• Step 2: Transcription
• Step 3: Translation
transcription
translation
What is RNA and why is it needed?
• RNA stands for RiboNucleic Acid. It is similar in structure to
DNA, but has a few differences that make it a necessary
component for transcription and translation to occur.
DNA
RNA
Double (2)
Single (1)
Deoxyribose
Ribose
Location
Nucleus
Starts in the nucleus and exits
Nitrogen Bases
A, T, G, C
Number of strands
Type of Sugar
Enzyme Used
Types
A, U, G, C
U = uracil
DNA Polymerase
RNA Polymerase
Only 1
Multiple types:
mRNA = messenger RNA
tRNA = transfer RNA
rRNA = ribosomal RNA
Types of RNA
– Messenger RNA (mRNA) carries the message that will be translated to
form a protein. (messenger)
– Transfer RNA (tRNA) brings amino acids from the cytoplasm to a
ribosome. (taxi)
– Ribosomal RNA (rRNA) forms part of ribosomes where proteins are
made.
Transcription – The process that copies the genetic
code in DNA onto a strand of mRNA.
• Steps:
1.) Initiation – RNA polymerase attaches to the promotor, which is a
nucleotide sequence that marks where RNA polymerase should start
transcribing AND which DNA strand to transcribe.
2.) Elongation– The enzyme RNA polymerase reads the DNA code and
helps assemble a growing (elongating) mRNA molecule. As the mRNA stand
peels away from its DNA template, the two separated DNA strands will come
back together to reassemble the double helix.
3.) Termination – The RNA polymerase reaches a sequences of
nucleotides in the DNA template called a terminator. This sequences the
end of a gene, and RNA polymerase detaches from the mRNA and the
gene. mRNA leaves the nucleus and travels to the ribosome.
Follow the base pair rules, but remember that RNA does NOT have thymine
instead it has uracil!
DNA sequence =
mRNA sequence =
A T G G C T A A T
U A C C G A U U A
RNA Processing in Eukaryotic Cells
• In eukaryotic cells, mRNA must be modified and
processed in several ways prior to translation.
• 1st – A small cap (a single G nucleotide) at one end and a
long tail (a chain of 50-250 A’s called a poly-A tail) at the
other end are added the strand of mRNA.
• The cap and tail aid in the export of the mRNA through the
nuclear pores. They protect the mRNA from being attacking
by cellular enzymes, and help ribosomes bind to the mRNA
during translation.
• 2nd – Internal noncoding regions, called introns
(intervening sequences), are removed in a process called
RNA splicing. Exons, or expressed regions of mRNA,
remain and are sent out of the nuclues to be translated.
RNA Processing in Eukaryotic Cells
Transcription
Translation
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Translation is the process in which the messenger RNA (mRNA) molecule is
translated into a strand of amino acids (polypeptide chain = protein).
Translation converts mRNA messages into polypeptides or proteins. Translation
occurs in the cytoplasm at ribosomes!
A codon is a sequence of three nucleotides of mRNA that codes for an amino acid.
codon for
methionine (Met)
codon for
leucine (Leu)
Codon Chart
The genetic code matches each RNA codon with its amino acid or function.
tRNA
• An anticodon is a set of three nucleotides of tRNA that is
complementary to a mRNA codon. An anticodon is located on
a tRNA molecule.
Phases of Translation
1.) Initiation: Translation is signaled to begin.
• A mRNA molecule binds to a small ribosomal subunit. A special initiator tRNA binds
to a mRNA start codon (AUG) and brings the amino acid methionine and signals the
ribosome to start assembling a polypeptide.
• Next a large ribosomal subunit binds to the small one, creating a functional
ribosome. The initiator tRNA fits into one of the two tRNA-binding sites on the
ribosome. This site, called the P site, will hold the growing polypeptide. The other
tRNA-binding site, called the A site, is vacant and ready for the next amino-acidbearing tRNA molecule.
Important Note: Each amino acid is joined the correct tRNA molecule by a specific
enzyme. This process requires energy in the form of ATP.
2.) Elongation: Amino acids are added to the growing polypeptide one at a time.
• Codon recognition – tRNA anticodon pairs with mRNA codon at the A site.
• Peptide bond formation – a peptide bond forms b/w the previous amino acid and the incoming amino
acid attached to the tRNA
• Translocation – the tRNA at the P site leaves and the tRNA located at the A site translocates, or moves,
into the newly vacant P site.
• Elongation continues until a stop codon reaches the ribosome’s A site.
3.) Termination: A stop codon – UAA, UAG, or UGA – will signal to stop translation. The
polypeptide is then released and exits the ribosome.
How do mutations impact phenotype?
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Genetic mutations alter or change the DNA sequence in a chromosome. They
arise due to errors in DNA replication or recombination. They can also arise from
exposure to mutagens such as x-rays and UV light. The following are types of gene
mutations that may or MAY NOT affect the phenotype (physical appearance) of an
organism:
– Point mutation – A single-base is copied wrong and results in a different
nucleotide sequence and POSSIBLY a different amino acid sequence and
protein. This type of mutation is sometimes referred to as a base substitution.
– There are a number of mutations that are considered point mutations. They
include:
• Silent mutations – there is NO change in amino acid sequence or the type
of protein assembled.
• Missense mutations – there IS a change in amino acid sequence AND the
type of protein assembled.
• Nonsense mutations - there IS a change in amino acid sequence and it
results in a STOP codon stopping the formation of the protein.
– Frame-shift mutation – The addition or removal (insertion or deletion) of one
or more nucleotides which causes a shift in the reading frame of the sequence
results in a different amino acid sequence and therefore makes a different
protein.
Point Mutation vs. Frameshift
Mutation
mutated
base
Viral Reproduction & Protein Synthesis
• Viruses consist of a nucleic acid (DNA or RNA)
surrounded by a protein coat.
– Extremely small (smaller than a ribosome)
– Genetic material can be any of the following:
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Double-stranded DNA
Single-stranded DNA
Double-stranded RNA
Single-stranded RNA
– Genetic material is protected by a protein coat called
a capsid.
– Some viruses may have a viral (membranous)
envelope that surrounds the capsid and helps the
virus infect its host.
Viruses
• Viruses are host specific and have a limited host range. This means they can
infect only a very limited range of hosts.
• Viruses can infect all types of cells including bacteria, animals, plants, fungi,
and protists.
• Viruses come in a variety of shapes and sizes.
• Viruses can only reproduce within a host. The host cell provides the necessary
components for replication, transcription and translation of the viral genetic
material.
• Viruses that infect bacterial cells are called bacteriophages (A.K.A. phages).
– Two types of reproductive cycles can occur in bacteria.
• Lytic cycles
• Lysogenic cycles
Lytic vs. Lysogenic Viral Infections
• Lytic cycles: results in the death
of the host cell due to lysis
(rupture).
– A bacteriophage injects its DNA
into a host cell and takes over
the host components in order to
synthesize new copies of viral
DNA and capsids (protein
coats). As more and more
viruses are assembled within
the host, eventually the host
cell will rupture releasing
multiple new viruses.
– Examples of viruses that
undergo lytic cycles are the
viruses that cause:
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Influenza
SARS
Common cold
Rabies
• Lysogenic cycles: results in viral DNA
being passed on to each newly
replicated bacterial cell by way of a
prophage, which is simply viral DNA
that has been inserted into the
bacterial DNA.
• Viruses that reproduce using a
lysogenic cycle may appear to
be dormant or inactive because
they don’t cause host cell death,
unless they are exposed to a
change in environmental
conditions which may cause
them to enter into a lytic cycle.
• Example of viruses that undergo
lysogenic cycles are the viruses
that cause:
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Herpes
Hepatitis
Lytic vs. Lysogenic Cycles
Viral Genetic Material
• Diseases caused by DNA
viruses include:
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Hepatitis
Chicken pox
Herpes
DNA  RNA  protein
• Diseases caused by RNA
viruses include:
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Common cold
Measles
Mumps
AIDS
Polio
RNA  protein OR
RNA  DNA  RNA 
proteins (retroviruses)
• Uses reverse transcriptase
• AIDS
• Viruses are able to target specific
hosts because of the glycoproteins
found on the membranous
envelope around the capsid of
some viruses. The glycoproteins aid
in the identification and
recognition of specific cells.
• Vaccines are the best combat for
these viruses since vaccines expose
host cells to weakened or dead
forms of the virus, so that host
cells can make antibodies in
preparation for potential infection
of healthy viruses.
Emerging Viruses
• Viruses that appear suddenly or have recently come to
the attention of medical scientists are called emerging
viruses.
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HIV (human immunodeficiency virus)
Ebola
West Nile
SARS (severe acute respiratory syndrome)
• Emerging viruses form due to :
– Mutations (RNA viruses don’t have proofreading
capabilities…therefore more mutations)
– Contact between species
– Spread from isolated populations
Viruses, Viroids and Prions – Pathogenic
Problems for Animals and Plants
• Viroids – circular RNA molecules that infect
plants. They cause errors in the regulatory
systems that control plant growth.
• Prions – misfolded, infectious proteins that
cause misfolding of normal proteins in
animals. They are responsible for mad cow
disease (cows) and Creutzfeldt-Jakob disease
(humans).
Bacteria – Transfer of DNA
• A bacterial cell’s genotype and phenotype can be
altered relatively easily by incorporating new
genetic material from another source.
• DNA can be transferred between bacterial cells in
one of three ways, all of which increase genetic
variation:
– Transformation – uptake of foreign or naked DNA
from the surrounding environment occurs
– Transduction – uptake of foreign bacterial DNA by
transmission from a bacteriophage (virus)
– Conjugation – bacteria exchange DNA directly through
the use of conjugation pili
Bacterial DNA Transfer Methods
Regulation of Gene Expression in Prokaryotes
• Bacteria often respond to environmental change by
regulating transcription.
– In bacteria (prokaryotes) genes are often clustered into units
called operons.
– An operon consists of three parts:
1)
2)
3)
Operator: controls the access of RNA polymerase to the genes on a
strand of DNA. Think of the operator as the ON/OFF switch.
Promoter: site on the DNA template where RNA polymerase
attaches.
Genes of the Operon: This is the entire stretch of DNA required for
all the enzymes produced by the operon.
Operons
• Operons are typically turned “on”.
• Operons can be switched off by a protein called a repressor that binds to
the operator of an operon preventing RNA polymerase from binding to
promoter.
• Regulatory genes located near an operon can code for the production of
repressor proteins.
• Types of operons:
– Repressible operon: The binding of a specific repressor protein to the
operator shuts off transcription. If the molecule being produced by the
operon is present in the cell, that molecule can act as a corepressor. A
corepressor is a molecule that binds to the repressor and helps to activate the
repressor. The repressor that binds to the operator of the operon and turns
OFF the gene. A repressible operon is normally anabolic, building an essential
organic molecule.
• Ex) trp operon – tryptophan acts as corepressor.
– Inducible operon: Binding of an inducer to the repressor inactivates the
repressor and turns on transcription. An inducer is a small molecule that binds
to and inactivates the repressor protein. Since the repressor is inactivated, it
CANNOT bind to the operator and therefore CANNOT block RNA polymerase.
An inducible operon is normally catabolic, breaking down food molecules for
energy.
• Ex) lac operon – the inducer in the lac operon is allolactose.
lac operon
trp operon
Negative vs. Positive Control of Gene
Expression in Prokaryotes and Viruses
• Negative Control – regulatory proteins
(repressors) inhibit gene expression by binding to
DNA and blocking transcription.
• Positive Control – regulatory proteins (inducers)
stimulate gene expression by binding to DNA and
stimulating transcription.
• Certain genes are continuously expressed; that is,
they are always turned “on”
– Ex) ribosomal genes or any gene needed ALL the time
Gene Regulation in Eukaryotes
• In eukaryotes all somatic cells of an organism contain the
same genome, therefore cells express only the genes they
need to carry out a specific function in a process called
differential gene expression. This type of gene expression
leads to cell differentiation, or the development of cells
with different functions
• The tightly packed condensing of DNA to form
chromosomes can inhibit transcription because it makes it
more difficult for RNA polymerase to bind to the promoter
of a gene.
– DNA methylation – the addition of methyl groups to DNA
causes the DNA to be more tightly packaged; prevents
transcription and gene expression
– Histone acetylation – acetyl groups are added to amino acids of
histone proteins and makes chromatin less tightly packed;
increases transcription and gene expression
Gene Regulation in Eukaryotes
• Eukaryotic genes are NOT found as operons.
• Eukaryotic genes contain the following:
– Promoter: A DNA sequence where RNA polymerase binds
and starts transcription
– Transcription factors: types of proteins that aid in
transcription (2 types)
1st - Proteins that recognize and bind to the promoter.
2nd - Specific transcription factors called activators bind to the
enhancer.
– Enhancer: DNA sequence that binds with an activator,
which causes the DNA to loop so that the transcription
factors on the promoter can bind with the transcription
factors (activators) on the enhancer sequence, therefore
initiating transcription.
– Terminator: DNA sequence that stops transcription
Transcription Factor Complex
Cancer Regulatory Genes
• Proto-oncogenes: genes that code for proteins that are
responsible for normal cell growth
– Proto-oncogenes can become oncogenes (cancer causing genes)
if mutated.
• Tumor-suppressor genes: genes that help inhibit cell
division.
– Cancer can arise if tumor-suppressor genes are repressed or
turned OFF.
– P53: An important tumor-suppressor gene. The p53 gene
produces a protein that suppresses cancer in more than one
way:
• The p53 protein can activate the p21 gene, whose product halts the
cell cycle by binding to cyclin-dependent kinases, allowing time for
DNA repairs to be made.
• The p53 protein can turn ON genes directly involved in DNA repair.
• When DNA damage is too great to repair, the p53 protein activates
“suicide” genes whose products cause cell death or apoptosis.
What do we do with this knowledge?….
Biotechnology & Genetic Engineering!
• Biotechnology – Process of manipulating organisms or their components for
the purpose of making useful products. The term is commonly associated with
genetic engineering. Genetic engineering is the process of manipulating genes
and genomes.
• Genetic engineering has impacted the fields of medicine, forensics and
agriculture.
• The following are examples of biotechnology/genetic engineering:
– Selective breeding – The process of breeding organisms that results in offspring
with desired genetic traits.
– Gene splicing – A type of gene recombination in which the DNA is intentionally
broken and recombined using lab techniques.
• Recombinant DNA: DNA that has been artificially made using DNA from different sources
– Genetically modified organisms – An organism whose genetic material has been
altered through some type of genetic engineering technology.
– Gene therapy – The intentional insertion, alteration, or deletion of genes within an
individual’s cells and tissues for the purpose of treating a disease.
– PCR (Polymerase Chain Reaction) – A method used to amplify DNA without the use
of a cell, especially if the source is impure (DNA left at a crime scene).
PCR (Polymerase Chain Reaction)
• It can make thousands to millions of copies of a DNA fragment
so further molecular studies can be conducted.
Three steps:
• Denaturation:
Heat briefly to
separate DNA
strands.
• Annealing: Cool
to allow primers
to form hydrogen
bonds with ends
of target
sequences.
• Extension: DNA
polymerase adds
nucleotides to
the 3’ end of
each primer.
Gene Splicing – Formation of Recombinant
DNA by TRANSFORMATION
DNA Microarray Assays
A technique that allows a
genome-wide study of gene
expression in organisms.
1st – Small amounts of singlestranded DNA fragments
representing different genes are
fixed to a glass slide in a tight
grid called a DNA chip.
2nd – mRNA molecules from the
cells being tested are isolated
and converted to cDNA by using
reverse transcriptase, then
tagged with a fluorescent dye.
3rd – The cDNA bonds to the
ssDNA on the chip, indicating
which genes are “on” in the
cell. This allows researchers to
see differences in gene
expression to help in the
identification of diseases.
Cloning
– Cloning: A process in which DNA, a cell or an organism is copied from an
original source, therefore resulting in an identical piece of DNA, cells or
organisms.
• Types of cloning:
1.) Gene cloning: scientists produce multiple copies of specific segments
of DNA that they can then work with in the lab.
2.) Reproductive cloning: identical organisms are created
3.) Therapeutic cloning: identical cells (stem cells) are created
How has genetic engineering
impacted…Forensics?
• Forensics: The science of tests and techniques used during
the investigation of crimes.
– Restriction Fragment Length Polymorphisms (RFLPs)
– DNA Fingerprinting
– DNA Gel Electrophoresis
How is genetic engineering
impacting...Medicine?
• Gene Therapy
• Gene Cloning
• Gene Splicing
How is genetic engineering
impacting…Agriculture?
• Genetically Modified
Organisms (GMO)
• Selective Breeding
• Gene Splicing
• Cloning