Download Topic # 7: Nucleic Acids

Topic # 7:
Nucleic Acids
I. DNA Structure and replication
A. Semi-conservative replication of DNA
1. The replication of DNA is semiconservative and depends on the
complementary bases pairing
2. When a cell prepares to divide, the two
strands of the double helix separate
3. Each old strand serves as a template to
build a new strand  two new molecules
form
4. The new strands form by adding
nucleotides, one by one and linking them
together
I. DNA Structure and replication
5. Each new DNA strand is made up of half a
new strand and half of an old strand
 semi-conservative
6. complementary base pairing
a. When the strands are open,
nucleotides
are exposed
b. Adenine will attract free Thymine
Cytosine will attract free Guanine
Thymine will attract free Adenine
Guaning will attract free Cytosine
c. Enzymes direct this process
II. The Hershey Chase Experiment
A. Alfred Hershey and Martha Chase
1. Wanted to determine if DNA or proteins
were the genetic material
2. Used viruses to perform the experiments
3. Viruses are made up of proteins on the
outside and DNA on the inside
4. The T4 bacteriophage was used for the
research
5. They added a radioactive marker to the
protein on the T4bacteriophage
6. No radioactivity in the resulting culture
7. They then added a radioactive marker to
II. The Hershey Chase Experiment
DNA of the T4 bacteriophage
8. The resulting culture contained the
radioactivity
III. DNA Structure and replication
A. The Watson and Crick model suggested
semi-conservative replication
1. DNA structure suggested a mechanism for
DNA replication
2. Several lines of evidence came together
to
lead to the knowledge of the structure of
DNA
III. DNA structure and replication
a. Linus Pauling: molecular modeling
b. Rosalind Franklin: X-ray diffraction and
careful examination of her
photographs
c. Erwin Chargaff: base composition
studies
3. Watson and Crick
a. First models had sugar-phosphate
strands wrapped around each other
b. nitrogen bases were facing outward
c. Franklin countered that the nitrogen
bases would face the center of the helix
III. DNA structure and replication
4. Franklin’s X-Ray diffraction showed that
the DNA helix was tightly packed
5. The tight packing could only occur if a
purine paired with a pyrimidine and if their
bases were upside down
a. A-T, T-A
b. C-G, G-C
6. This pairing suggests the mechanism by
which DNA replication could occur
B. The role of nucleosomes in DNA packing
1. Nucleosomes help to supercoil DNA
2. DNA is associated with proteins - histone
III. DNA structure and replication
3. Histones package the DNA into structures
called nucleosomes
a. Central core of 8 histone molecules
b. DNA wrapped around it
c. An additional histone (the H1 histone)
holds the DNA in place
III. DNA structure and replication
B. The leading strand and the lagging strand
1. DNA replication is continuous on the
leading strand and discontinuous on the
lagging strand
2. The two strands are antiparallel
3. The leading strand is synthesized
continuously
4. The lagging strand is made in fragments
moving away from the replication fork
5. These fragments are called Okazaki
fragments
III. DNA structure and replication
C. Proteins involved in replication
1. DNA replication is carried out by a complex
system of enzymes
2. DNA helicase unwinds the DNA at the
replication fork
3. Topoisomerase releases the strain that
develops ahead of the helicase
4. Single-stranded binding proteins keep the
strands apart long enough to allow the
template strand to be copied
5. RNA primer
a. Many RNA primers are on the lagging
III. DNA structure and replication
strand, but only one is needed on the
leading strand
b. RNA primers are formed by RNA
primase
c. They are 10-12 nucleotides long
d. The primers are sequenced according to
the part of the strand where they are
placed
e. RNA primers are necessary to initiate
the activity of DNA polymerase
C. DNA polymerase
1. Covalently links the nucleotide to the 3’
III. DNA structure and replication
a. Proof-reading
b. Polymerization
c. Removal of RNA primers as they are no
longer needed
D. The direction of replication
1. DNA polymerases can only add nucleotides
to the 3’end of a primer
2. Prokaryotes have only one origin of
replication
3. Eukaryotes have many different origins
4. Replication occurs in both directions away
from the origin
III. DNA structure and replication
5. The PO4-3 of the new DNA nucleotide is
added to the 3’ carbon of the deoxyribose of
the nucleotide at the end of the chain
6. Therefore, DNA replication occurs in a
5’3’ direction
III. DNA structure and replication
E. Non-coding regions of DNA have important
functions
1. Some regions of DNA do not code for
proteins but have other important functions
2. DNA is used as a guide for the production
of polypeptides using the genetic code
3. not all of DNA codes for polypeptides
4. some code for production of tRNA or rRNA
5. some regulate gene expression
6. the majority of the eukaryotic genome is
non-coding
7. repetitive sequences can be common
III. DNA structure and replication
8. telomeres
a. Repetitive sequences at the end of a
chromosome
b. Serves a protective function for the
chromosome
i. The enzymes that replicate DNA
cannot continue replication all the way
to the end of the chromosome
ii. The DNA sacrifices the repetitive
telomere portion each time the cell
divides  protects the genes
IV. Transcription and gene expression
A. Regulation of gene expression by proteins
1. Gene expression is regulated by proteins
that bind to specific base sequences in
DNA
2. Some proteins are always necessary for
the survival of an organism
3. These are expressed in an unregulated
fashion
4. Other proteins need to be produced at
certain times and in certain amounts so
their expression needs to be regulated
5. In prokaryotes, it happens as a result of
IV. Transcription and gene expression
environmental factors
Ex. E. coli can only produce the enzyme
needed to break down lactose after the
disaccharide has been absorbed
6. In eukaryotes, genes are also regulated in
response to environmental conditions as well
7. There are proteins that regulate transcription
when they bind to DNA
a. Enhancers
b. Silencers
c. Promoter-proximal elements  nearer
to the promoter and binding of proteins
IV. Transcription and gene expression
B. The impact of the environment on gene
expression
1. The environment of a cell and of an
organism has an impact on gene expression
2. “nature vs nuture” debate
3. Twin studies have been done to see what
phenotypes are expressed and if they can be
attributed to environment or to heredity
4. For some traits, the influence of the
environment is unequivocal
a. Skin pigmentation during sun exposure
5. embryonic development
IV. Transcription and gene expression
a. Embryos contain uneven distribution of
chemicals called morphogens
b. Concentration of morphogens affect
gene expression
c. Produce different fates for the
embryonic cells
i. Dependent on their position in the
embryo
d. Coat color in cats
i. “C” gene codes for the enzyme
tyrosinase – the 1st step in pigment
production
IV. Transcription and gene expression
ii. “c” is a mutant allele that allows
for
normal pigment production at temps
below body temperature
C. Nucleosomes regulate transcription
1. Nucleosomes help to regulate transcription
in eukaryotes
2. eukaryotic DNA is associated with histones
3. chemical modification of the tails of
histones help to determine if a gene will be
expressed or not
a. Acetyl group
c. Phosphate group
b. Methyl group
IV. Transcription and gene expression
D. The direction of transcription
1. Transcription occurs in a 5’ to 3’ direction
2. Production of mRNA occurs in three stages
a. Initiation
b. Elongation
c. Termination
3. Begins near a site in DNA called the
promoter
4. Once binding of RNA polymerase occurs,
the DNA is unwound by the RNA
polymerase forming an open complex
5. RNA polymerase slides along the DNA
IV. Transcription and gene expression
6. synthesizes a single strand of RNA
An animation of the process…
E. Post-transcriptional modification
1. Eukaryotic cells modify mRNA after
transcription
2. Transcription occurs in the nucleus and
modification afterward also occurs there
before it leaves the nucleus
3. Introns are removed from the RNA
introns  intervening sequences…they
intervene with the genetic sequence
IV. Transcription and gene expression
4. Exons are left behind
 the expressed portion of the gene
F. mRNA splicing
1. Splicing of mRNA increases the number of
different proteins an organism can produce
2. alternative splicing is a process during gene
expression whereby a single gene codes for
multiple proteins
3. a particular exon may or may not be
included in the final mRNA
4. So  proteins translated from
alternatively spliced mRNAs will differ in their
IV. Transcription and gene expression
4. Exons are left behind
 the expressed portion of the gene
F. mRNA splicing
1. Splicing of mRNA increases the number of
different proteins an organism can produce
2. alternative splicing is a process during gene
expression whereby a single gene codes for
multiple proteins
3. a particular exon may or may not be
included in the final mRNA
4. So  proteins translated from
alternatively spliced mRNAs will differ in their
V. Translation
A. The structure of the ribosome
1. The use of molecular visualization software
to analyze the structure of eukaryotic
ribosomes and a tRNA molecule
2. Ribosome structure includes
a. proteins and ribosomal molecules
(rRNA)
b. two sub-units, one large and one small
c. Three binding sites for tRNA on the
surface of the ribosome – two tRNA
molecules can bind at the same time to the
same ribosome
V. Translation
surface of the ribosome
e. Each ribosome has three tRNA binding
sites
i. E – exit site
ii. P – peptidyl site
iii. A – amino acyl site
3. All tRNA molecules have
a. Sections that become double-stranded
by base pairing, creating loops
b. A triplet of bases called the anticodon
which is part of a loop of seven unpaired
bases
V. Translation
c. Two other loops
d. The base sequence CCA at the 3’ end
which forms a site for attaching an amino
acid
4. tRNA-activating enzymes
a. tRNA-activating enzymes illustrate
enzyme-substrate specificity and the role of
phosphorylation
b. Each tRNA molecule is recognized by a
tRNA-activating enzyme that attaches a
specific amino acid to the tRNA, using ATP
for energy
V. Translation
c. 20 different amino acids, so 20 different
tRNA molecules (at least)
d. The active site of the activating enzyme
is specific to both the correct amino acid
and the correct tRNA
e. Energy from ATP is needed for the
attachment of amino acids
f. Once ATP and amino acid are attached
to
the active site, the amino acid is
activated
g. The activated amino acid is covalently
attached to the tRNA
V. Translation
to the growing polypeptide
B. Initiation of translation
1. Initiation of translation involves assembly
of the components that carry out the process
2. to begin translation, mRNA binds to the
small ribosomal subunit at an mRNA binding
site
3. An initiator tRNA carrying methionine then
binds at the start codon “AUG”
4. The large ribosomal subunit then bind to
the small one
5. The initiator tRNA is in the P site
V. Translation
6. The next codon signals another tRNA to
bind
7. a peptide bond is formed between the
amino acids in the P and the A site
C. Elongation of the polypeptide
1. Synthesis of the polypeptide involves a
repeated cycle of events
2. following initiation, elongation occurs
through a series of repeated steps
3. the ribosome translocates three bases
along the mRNA, moving the tRNA in the P
site to the E site
V. Translation
6. The next codon signals another tRNA to
bind
7. a peptide bond is formed between the
amino acids in the P and the A site
C. Elongation of the polypeptide
1. Synthesis of the polypeptide involves a
repeated cycle of events
2. following initiation, elongation occurs
through a series of repeated steps
3. the ribosome translocates three bases
along the mRNA, moving the tRNA in the P
site to the E site
V. Translation
4. this allows a tRNA with the appropriate
anticodon to bind to the next codon
5. this vacates the A site
6. next amino acid can bind in the same way
…on to the animations!!!
D. Termination of translation
1. Disassembly of the components follows
termination of translation
2. A stop codon is reached and the free
polypeptide is released
V. Translation
3. movement of translation is from the 5’ to
3’
end of the mRNA strand
4. Stop codons
i. UAG, UAA, UGA
***SO!! Replication, Transcription and Translation
ALL occur in a 5’ to 3’ direction” ***
E. Free ribosomes
1. Free ribosomes synthesize proteins for use
primarily in the cell
2. these are proteins destined for use in the
cytoplasm, mitochondria and chloroplasts
V. Translation
F. Bound ribosomes
1. Bound ribosomes synthesize proteins
primarily for secretion or for use in lysosomes
2. proteins must be sorted so they end up in
their correct location
3. Proteins that are destined for use in the
ER,
the Golgi apparatus, lysosomes, the plasma
membrane or outside the cell are
synthesized
by ribosomes bound to the ER
G. The coupling of transcription and translation
in prokaryotes
1. Translation can occur immediately after
V. Translation
transcription in prokaryotes due to the
absence of a nuclear membrane
2. in eukaryotes, cellular functions are
compartmentalized
3. in prokaryotes, they are NOT
4. once transcription is complete in
eukaryotes, the transcript is modified before
exiting the nucleus
5. In prokaryotes, as soon as the mRNA is
transcribed, translation begins
H. Primary Structure
1. The sequence and number of amino acids
V. Translation
the polypeptide is the primary structure
2. a chain of amino acids is called a
polypeptide
3. The sequence is the primary structure
I. Secondary structure
1. the secondary structure is the formation of
alpha helices and beta pleated sheets
stabilized by hydrogen bonding
2. yeah…there’s a picture on the next slide
J. Tertiary structure
1. the tertiary structure is the further folding
of the polypeptide stabilized by interactions
Bonds form
between the
carboxyl and the
amino group for
both parts of the
secondary
structure
V. Translation
2. three dimensional shape of the protein
3. it’s the result of the interaction of R-groups
with one another
4. Several types
a. Positively charged R-groups interact
with negatively charged R-groups
b. Hydrophobic amino acids will orient
themselves toward the center of the
polypeptide to avoid contact with water
while hydrophilic amino acids will orient
themselves outward
c. Polar R groups will form hydrogen
V. Translation
with other polar R groups
d. The R-group of the amino acid cysteine
can form a covalent bond with the Rgroup of another cysteine forming what is
called a disulfide bridge
K. Quartenary structure
1. The quartenary structure exists in proteins
with more than one polypeptide chain
2. Insulin is formed from two polypeptide
chains
3. Hemoglobin is formed by four polypeptide
chains! FOUR!
V. Translation
4. The biological activity is related to its
primary, secondary, tertiary and quaternary
structure
5. certain treatments can alter the structure
permanently and change how the protein
functions  denaturing the protein
i. pH changes
ii. Temperature extremes