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Chapter 10 – Molecular Biology of the Gene
The structure of the Genetic Material
I. Intro
A. Viruses – basically packaged nucleic acid particles
1. Living or Non-living
a) genetic material is nucleic acid,
(1) not cellular
(2) does not reproduce on its own.
b) Nucleic acid wrapped in a protein coat (capsid)
and
c) capsid and nucleic acid is sometimes surrounded
by a membranous envelope (phospholipids +
membrane proteins)
d) They are tiny (largest are 200nm – 1/100th of a
human cell) and
(1) do not carry the tools they need to reproduce
(2) they need the cell’s tools!
2. Brief life cycle of Herpevirus (similar to other viruses)
a) Viruses Gain access to specific cells (nerve cells in
this case)
(1) trick it - proteins on virus (the ligand) fit into and
bind with natural cell receptors – structure function.
(2) cell takes up virus, unknowingly
b) viral DNA enters the nucleus
c) It can remain dormant until the time is right
d) Then hijack the cell and use the cellular machinery
for its own purpose (to reproduce of course…).
e) Cell fills with virus particles and lyses, releasing
the particles to infect other cells (the sores).
3. Once infected, it remains permanently latent,
integrated into the nerve cell’s DNA.
4. 75% Americans carry HSV-1 and 20% have HSV-2
5. many infected people never show symptoms
6. ability to remain latent is somewhat unusual, a trait
shared with HIV
7. Viruses are far simpler on the molecular level
compared to Mendel’s peas and Morgan’s flies
a) because of this, virus gave us our first glimpse of
how DNA controls heredity – molecular biology
B. The chromosomal theory of inheritance set the stage for
the development of a molecular understanding of the gene
C. Remember, we still don’t know what molecule(s) is
responsible for heredity. We know it has to do with
chromosomes, and we know it is made of protein and DNA.
So which is it? Scientists thought protein.
II. How was DNA determined to be the genetic
(hereditary) material of life?
A. Began in 1928 – English bacteriologist Frederick Griffith
1. He heat-killed a pneumonia-causing bacteria
Streptococcus pneumonia (smooth strain)
2. Took some substance from the dead bacteria and
gave it to a harmless form of the bacteria (rough strain)
3. The rough strain and its descendents were
transformed into the infectious smooth strain!
4. What was this “transforming factor”? Griffith and
everyone else believed it to be protein!
5.
Inject:
Rough strain
Smooth strain
Heat-killed
smooth strain
Rough strain +
heat-killed
smooth strain
Outcome:
Mouse lives
Mouse dies
Mouse lives
Mouse dies
B. Chromosomes were known to be composed of:
1. Protein – more complex – 20 amino acids – therefore
they believed it to be the genetic material
2. DNA
C. Oswald Avery continued Griffith’s experiments
1. separated the different components of the heat-killed
smooth Streptococcus pneumonia into the 4 major
macromolecules: polysaccharides, proteins, nucleic
acids, and lipids.
2. mixed each component separately with the rough
Streptococcus pneumonia and injected each into mice.
3. Nucleic acid was the lethal mixture
4.
Inject:
Rough strain +
lipids
Rough strain +
carbs
Rough strain +
proteins
Rough strain +
nucleic acid
Outcome:
Mouse lives
Mouse lives
Mouse lives
Mouse dies!!
5. So is it DNA or RNA?
6. He treated the nucleic acid with an enzyme that
hydrolyzes RNA (RNase), added it to the Rough
Streptococcus pneumonia and it killed the mice…DNA
was it!!
Inject:
Rough strain +
nucleic acid +
RNase
Outcome:
Mouse dies!!
D. Hershey and Chase experiment – 1952 – confirmed DNA
to be hereditary material.
1. Studied T2 bacteriophages
a) bacteriophages (bacteria eaters) are bacterial
viruses – made of protein and DNA
2. They knew the T2 could take over bacterial cells, they
wanted to confirm that is was DNA doing this and not
protein.
3. The experiment (Fig. 10.1AB):
a) Grew phage in different radioactive elements
b) radioactive Sufur to label protein (S only in the
proteins), radioactive phosphorous to label DNA
(phosphorus only found in the DNA).
c) use phage to infect bacteria (E. coli)
d) blend to dislodge any phage material stuck to
outside of cell
e) centrifuge cells down and look for radioactivity in
the cells
f) phage with labeled DNA resulted in radioactive
cells. Thus, DNA was being injected into the bacteria.
g) return bacteria to growth media (liquid that
bacteria grow in) and they lyse, releasing virus with
radioactive DNA, but not protein.
4. Conclusions –
a) only DNA is injected, protein left outside
b) it is the injected DNA that cause the cells to
produce additional phage
5. These results plus others gathered during the 1920’s
through the early 1950’s convinced the world that DNA
was the hereditary material.
6. This set the stage for one of the most controversial
and celebrated quests in all of science, the structure of
DNA!
III. DNA and RNA are polymers of nucleotides
A. By 1952, scientists knew a great deal about DNA
including all of its different atoms types and how they are
covalently bonded to each other.
B. What they did not know was the specific three
dimensional arrangement of these atoms that gave DNA its
unique properties
1. the ability to store genetic information
2. copy it
3. pass it to the next generation
C. nucleotides – monomers of nucleic acid – phosphate,
sugar, nitrogenous base
D. polynucleotide – polymer of nucleotides
E. sugar phosphate backbone - Nucleotides attach to each
other via the phosphate of one nucleotide and the sugar of
the next
F. DNA – DeoxyriboNucleic Acid
1. Deoxyribo- deoxyribose sugar
2. Nucleic – found in the nucleus
3. Acid – acidic phosphate group
G. 4 flavors of bases found in DNA – 2 groups
1. Purines (double-ring structure)
a) Adenine (A)
b) Guanine (G)
2. Pyrimidines (single ring structure)
a) Cytosine (C)
b) Thymine (T)
H. RNA – ribonucleic acid
1. Ribose sugar instead of deoxyribose (compare sugar
structures)
2. Phosphate is the same as DNA
3. RNA uses the same nitrogenous bases as DNA except
instead of Thymine (T) it uses Uracil (U) – show how
similar
4. Summary : Ribose instead of deoxyribose, U instead of
T, single stranded vs. double stranded
I. DNA is a double helix
1. Structural denotes function (hammer for nails) – so
learning the shape of DNA was critical to understanding
its role in heredity
2. The race for the structure of DNA was on!
3. The “winners”: James D. Watson (American) and
Francis Crick (English).
a) Science is not a solitary act – they had a great deal
of information like the structure of nucleotides,
Rosalind Franklin’s X-ray crystallographic pictures
of DNA – used to deduce the helical nature, Erwin
Chargaff’s data showing that the amount of A and T,
and C and G were always equal, and previous
knowledge that different species had different ratios
of A + T to G + C. – mention the book
4. The model that fit all the data was a double helix (a
twisted rope latter) with sugar-phosphate backbones as
the rope rails and the nitrogenous bases as the steps.
5. A always bonds with T and C always bonds with G –
called base pairs
6. No restriction on linear sequence of nucleotides
7. Structure was published in 1953 and led immediately
to the proposed mechanisms about DNA function.
8. James D. Watson was the Cold Spring Harbor
Laboratory Chancellor here on Long Island until October
14th 2007 when suspended from his responsibilities due
to comments he made that were published in the The
Sunday Times (U.K.).
DNA REPLICATION
IV.
DNA replication depends on specific base pairing
A. Complete and faithful copies of DNA must be produced
(replicated) during the cell cycle
B. Watson and Crick proposed a model for how DNA
replicates (is copied)
C. The mechanism proposed and confirmed at the end of
the 1950’s = semi-conservative model
1. each polynucleotide strand acts as a template on
which a new strand can be assembled according to base
pair rules (A-T, C-G).
D. Although it sounds simple, many proteins are involved.
1. DNA needs to be unwound, held open, and the proper
bases must be inserted one at a time by enzymes called
DNA polymerases over millions and millions of bases!!
E. The copy mechanism is VERY precise at a speed of 50
to 500 per second. There is a mistake on average of
1/1,000,000,000!
V. DNA replication: A closer look
A. ORIGINS OF REPLICATION (ORI)
1. many occur at same time in eukaryotes (speeds up
replication)
2. Replication bubble - forms at ORI – DNA is unwound
and unzipped.
3. replication goes in both directions
B. DNA polymerases – enzyme (protein) that synthesizes
DNA
1. Reminder: DNA is polar (different ends - there is a 3’
end and 5’ end)
2. DNA polymerase can only attach nucleotides to the 3’
end of a growing daughter strand
3. Therefore, replication proceeds 5’ to 3’
a) Leading strand – made continuously 5’ to 3’
b) Lagging Strand – made in short pieces (Okazaki
fragments) - “glued” together by DNA Ligase.
4. DNA polymerases can also proofread the new
daughter strands to see if they made a mistake
C. More Workers:
1. DNA helicase – enzyme that unzips the DNA
2. Why do the DNA strands stay separated and not just
zip back together? Single stranded binding proteins coat
the swingle strands and don’t allow it to.
3. DNA pol cannot start from scratch, it always needs an
RNA primer that is added by the enzyme Primase
4. Now there is RNA in the DNA? Don’t worry, primer is
replaced by DNA later by another type of DNA polymerase
(there are many types)
D. DNA replication ensures that the genetic information is
accurately copied and passed along to the daughter cells
VI. THE FLOW OF GENETIC INFORMATION FROM DNA
TO RNA TO PROTEIN
A. DNA = molecule basis of genotype
B. proteins = molecular basis for phenotype
C. 1940’s American geneticists George Beadle and Edward
Tatum – studying nutritional mutants of the mold
Neurospora.
1. Found that genetic mutants lacked single enzymes
needed to complete metabolic pathways
2. one gene-one enzyme hypothesis
3. extended to the one gene-one protein hypothesis and
later restricted to the one gene-one polypeptide
hypothesis
D. The flow of information in gene expression is from DNA
to RNA (transcription) to polypeptide (translation).
E. 80 to 90% of human DNA is referred to as “junk” DNA,
meaning we have no idea what it is used for. Only 1-2% of
our 3billion base pairs are genes!!
VII. The Codon
A. DNA = a parts list with the parts being the proteins
B. Written using its own alphabet of only four letters A, T,
G and C corresponding to the nucleotides.
C. The list can be read just like a shopping list, we just
needed to learn how to read it.
D. Letters of the DNA alphabet form words, always only 3
letters long (triplet code) called codons
E. Each codon corresponds to a specific amino acid.
1. How many amino acids are there?
2. Compare that to how many possible codons there are.
3. So why does DNA use 3 letter words and not 2 or 1?
F. These words form sentences of varying length called
genes of which there are 30,000+ in humans, 1000’s per
chromosome
G. Genes are stuck inside the nucleus, however, and
protein needs to be made in the cytoplasm
H. Transcribe a copy of the gene as a mRNA (it carries the
message), which can cross the nuclear envelope and enter
the cytoplasm.
VIII. The genetic code is the Rosetta stone of life
A. The Genetic code tells us how to decipher the nucleic
acid language
IX. Transcription - produces genetic messages in the
form of RNA
A. In prokaryotes, transcription and translation both occur
in the cytoplasm.
B. In eukaryotes,the transcribing of DNA to messenger
RNA occurs in the nucleus
C. It is similar to replication in that the two DNA strands are
unwound and unzipped.
D. Only one strand serves as the template
E. RNA nucleotides follow the same base pair rules as DNA
except that U pairs with A instead of T.
F. RNA polymerase – the enzyme that transcribes the gene
into mRNA by polymerizing the appropriate RNA
nucleotides
1. Synthesizes mRNA 5’ to 3’ just like DNA polymerase
G. 3 stages
1. Initiation – RNA polymerase binds to the promoter,
DNA unwinds, RNA synthesis begins – promoter tells
RNA polymerase which strand to transcribe
2. Elongation – RNA polymerase continues polymerizing
RNA nucleotides along the DNA template according to
base pairing rules. As the RNA strand is made, it peels
away from the DNA as the DNA winds up again.
3. Termination – RNA polymerase reaches a special
terminator sequence of DNA bases, detaches and mRNA
is released.
X. RNA processing
A. Messenger RNA (mRNA) – carries the message from
DNA to ribosome (translation machinery) outside the
nucleus.
B. The mRNA is processed BEFORE leaving the nucleus
1. Cap and tail are added to the mRNA
a) Protects mRNA from cellular enzymes that would
otherwise degrade it.
b) Helps ribosome to recognize the mRNA
2. Non-coding regions are removed (RNA splicing)
a) Most plant and animal genes have INTRONS and
EXONS
(1) Introns – non-coding regions that may be cut out
(2) Exons – coding regions used to make protein
(3) Introns are removed and exons are spiced
together in a process known as RNA splicing.
(4) Alternative splicing – splice different
combinations of exons to get different mRNAs – so
one gene actually can code for multiple polypeptides!
XI.
Translation
A. Translation – rewording of a message into a new
language (nucleic acid language  amino acid language)
1. Transfer RNA (tRNA)
a) the translator – converts nucleic acid language to
amino acid language.
b) How do cells make tRNA? There are tRNA genes –
no translation of course
c) Structure- Function of tRNAs
(1) Single strand of RNA, ~80 nucleotides
(2) Parts of a tRNA
(a) Folds upon itself to form double-stranded regions
(b) amino acid attachment site
(c) single-stranded loop with special triplet of bases
called an anti-codon.
(i) Anti-codon – complementary to the
mRNA codon
2. tRNA synthetase – enzyme that attaches the proper
amino acid to the proper tRNA to make an aminoacyl
tRNA
a) Amino acids are readily available in the cytoplasm
from recycled proteins, digested food, or biosynthetic
pathways.
B. Ribosomes
1. huge translation machine
2. Structure
a) composed of protein and rRNA (ribosomal RNA)
b) arranged in two massive subunits
(1) small subunit
(2) Large Subunit
c) There are two tRNA binding sites
(1) P site (peptidyl site) – binds the tRNA with the
growing peptide
(2) A site (aminoacyl site) – binds the tRNA with the
next amino acid to be added (aminoacyl tRNA)
3. Function
a) coordinates the mRNA, tRNA, and growing
peptide chain to allow synthesis.
C. Translation can be divided into 3 phases similar to
transcription
1. Initiation
a) small ribosomal subunit and appropriate tRNA
(MET attached and UAC anitcodon) binds to the start
(initiation) codon AUG
b) Large ribosomal subunit attaches placing initiator
tRNA in the P site.
2. elongation
a) codon recognition – anticodon of incoming tRNAamino acid complex binds with the codon at the
ribosome’s A site.
b) peptide bond formation – a polypeptide bond is
formed between the growing polypeptide (attached to
tRNA in P-site) and the new amino acid and the entire
chain is now on the A-site tRNA.
(1) Formation of the peptide bond is catalyzed by an
enzyme within the ribosome
c) translocation – The P-site tRNA leaves, the
ribosome slides over, the A-site tRNA-polypeptide
chain complex is now in P-site. - repeat back to step 1
3. Termination
a) elongation continues until a STOP CODON (UAA,
UAG, UGA) is reached.
b) release factor – a protein that binds stop codon in
the A site signaling the end
c) ribosome subunits fall off to find a new mRNA to
translate.
D. The polypeptide folds into its tertiary structure both
during and after translation.
E. After translation, a number of polypeptides may come
together to form a protein with quaternary structure.
XII. Review: The flow of genetic information in the cell
is DNA  RNA  protein
A. Transcription – synthesis of mRNA complementary to a
DNA template – in the nucleus
B. Translation – conversion of info within mRNA to a
polypeptide – in the cytoplasm
XIII. How can genes be altered?
A. Mutation – any change in the nucleotide sequence of
DNA
B. Two general flavors of mutation
1. Substitutions (point mutations) – change one base to
another
a) 3 possible outcomes
(1) may change the encoded amino acid resulting in
an abnormal gene product:
(a) Sickle Cell anemia – a single substitution in the
gene for hemoglobin (AT) resulting in a change in
the sixth amino acid from Glu to Val.
(2) The change may do nothing at all:
(a) If new codon still codes for the same amino acid
(silent mutation)
(b) If the change in amino acid does not affect the
function of the protein
(3) In rare cases, base substitutions lead to genes that
may enhance the success of the individual.
(a) Such mutations provide genetic variability that
MIGHT lead to the evolution of the species
2. Deletions and Insertions
a) Tend to be more severe – can result in a frame
shift, affects all of the amino acids downstream
(1) reading frame = the triplet grouping of codons
b) Will almost always result in a non-functional
polypeptide
C. Mutagenesis (formation of a mutation) can occur:
1. Spontaneously - Error in DNA replication or
recombination, or other mutations of unknown cause
2. Mutagens - physical (radiation) and chemical agents.
D. Mutagenesis might result in cancer (mutate genes
involved in halting the cell cycle – p53 – the “guardian
angel gene”)
1. can activate DNA repair proteins when DNA has
sustained damage.
2. It can hold the cell cycle at the G1 check point on DNA
damage recognition (if it holds the cell here for long
enough, the DNA repair proteins will have time to fix the
damage and the cell will be allowed to continue the cell
cycle.)
3. It can initiate apoptosis (programmed cell death) if the
DNA damage is irreparable.
E. While mutations are usually harmful, they are also
extremely useful:
1. Responsible for the rich diversity of genes in the
world, making evolution by natural selection possible
2. Essential tools for scientists –
a) Mendels naturally occurring mutations or
Morgans X-ray induced mutations generate different
alleles for study
b) Also important for understanding protein function
– change one amino acid to another and see how the
protein reacts
VIRUSES: PACKAGED GENES
A. Viruses are essentially nothing more than packaged
genes
B. All genes want is to reproduce themselves (otherwise
they would not be around today).
1. Viruses depend on their host cells for replication,
transcription, and translation just as computer viruses
depend on your computer.
XIV. Bacterial phages (viruses)
1. reproduce in two general ways:
a) Lytic cycle – phage invades host cell, hijacks it like
a bank robber with a list of demands! It immediately
tells the host to replicate the viral nucleic acid,
transcribe and translate its protein-coding genes,
assemble new viruses, and causes the host cell to lyse
(pop), releasing the new reproduced phages.
b) Lysogenic cycle – the phage DNA quietly enters the
cell and inserts sneakily into the host DNA by
recombination and becomes a prophage (the bank
robber gets a job at the bank first).
(1) When the host replicates, so does the viral DNA –
can go on for MANY generations
(2) Environmental cue will direct the prophage to
switch to the lytic cycle (hold up the bank).
XV. Animals Viruses and Disease
1. Organisms from all 5 kingdoms (3 domains) have
viruses that infect their cells
2. Viruses have evolved to use DNA or RNA as their
genetic material
3. Can be enveloped or not enveloped
4. Go over viral structure – envelope, glycoprotein
spikes, protein coated RNA
B. Enveloped RNA virus – Influenza, mumps (reproduce
outside the nucleus)
1. Go over reproductive cycle - Figure 10.18B
a) Envelope fuses and protein-coated RNA enters
cytoplasm
b) Enzymes remove protein coat
c) RNA strand is used as a template to make
complementary RNA strands
(1) Serve as mRNA for protein synthesis
(2) Serve as template to make new viral genome
d) New coat proteins assemble around new viral RNA
genomes
e) Virus leaves the cells cloaked in plasma membrane
specially coated with viral glycoprotein spikes – does
not necessarily lyse the cell
2. The entire process occurs in the cytoplasm (other
viruses might reproduce in the nucleus). Different viruses
have evolved different mechanisms.
C. Enveloped dsDNA virus - Herpes viruses (chickenpox,
shingles, mononucleosis, cold sores, and genital herpes) reproduce in the cells nucleus
1. Can insert their DNA into the cells DNA as a provirus
similar to a prophage in the lysogenic cycle
2. Different strains of the Herpes virus causes different
disease
D. Viruses that attack quickly repaired tissue are usually
recoverable through mitosis while non-repairable tissues
(eg. Nerves / polio virus) are much more severe
E. How can we stop viruses?
1. Vaccines are important in these more sever cases
because they prevent infection
2. Not to be confused with antibiotics, which are
molecules that attack bacteria
3. Anitviral development is SLOW because of how hard it
is to find a molecule that will kill the virus without killing
the host cells
F. Three major human viruses causing disease: influenza,
cold viruses, herpes viruses
XVI. Plant viruses are serious agricultural pests
A. Most are RNA viruses
B. Plants whose epidermal layer has been damaged by
wind, injury, chill, or insects are more susceptible
C. Insects, Farmers, Gardeners may spread these viruses
(pruning shears and other tools)
D. Infected plants may pass virus to offspring
E. There are no cures for most viral plant diseases.
Research has focused on prevention and selective
breeding of resistant varieties.
XVII. Emerging viruses threaten human health
A. Predicted by the theory of evolution – “we live in
evolutionary competition with microbes. There is no
guarantee that we will be the survivors” – Joshua
Lederberg, geneticist
B. Some viruses
1. HIV – identified in the 1980’s
2. FLU – every year
3. EBOLA – identified in 1978
4. HANTAVIRUS
C. New viruses can arise:
1. Through mutation of existing viruses – RNA viruses
have VERY high mutation rates – no proofreading during
replication! – Influenza
2. Spread of existing viruses to a new host species
a) ~75% of human diseases originated in other
animals
(1) Hanatavirus common in rodents
D. Viruses can spread like fire
1. HIV – unnoticed for decades before until blood
transfusion technology, affordable travel, sexual
promiscuity, and abuse of IV drugs enabled the virus to
spread.
XVIII.
The AIDS virus makes DNA on an RNA
template
A. HIV is the virus that causes the disease AIDS
1. HIV – Human Immunodeficiency Virus
2. AIDS – Acquired Immune Deficiency Syndrome
3. Both terms describe the effect on the body – kills
several kind of white blood cells (WBC’s)
B. HIV is a retrovirus
1. Retroviruses synthesize DNA from an RNA template
(retro = reverse)
2. Requires a special enzyme called reverse transcriptase
(transcription in the reverse direction)
C. The newly synthesized viral DNA can insert into the
hosts DNA making it a provirus
D. Review structure and behavior of HIV
XIX. Virus research and molecular genetics are
intertwined
A. Scientists have a love-hate relationship with viruses
1. Some loves
a) Hershey Chase Experiments on phage T2
concluding that DNA is the hereditary material
leading to Watson-Crick model of DNA
b) Viral reproduction has since helped show how
genetic information flows from DNA to protein
c) Gene delivery (gene therapy)
2. The hates
a) Disease