Chapter 14 DNA p' 277298

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Chapter 14 DNA p. 277-298 A. Discovery of DNA
as hereditary material 1. Hammerling 1930s,
hereditary material in same area as nucleus in
algae
2. Briggs and King 1952, hereditary material is
stored in nucleus of eukaryotic cells
3. Griffith Experiment 1928, showed that live
bacterial cells could be transformed by dead
ones, ie.e. that hereditary information was being
passe from dead cells to live ones, tranforming
them, called bacterial transformation or simply
transformation
4. Avery, Macleod, McCarthy 1943, showed the
transforming factor of Griffiths was DNA, but
many scientists were still convinced it was
protein, not DNA.
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5. Hershey (Alfred) and Chase (Martha!)
Experiment, 1952 showed that definitvely that
DNA and not protein was the genetic meterial by
differentially lableling viruses (bacteriophages)
and using them to infect bacteria. a. why it
worked used viruses called t-phages, which
consist only of DNA and protein. (see next trans)
b. used differential labeling c. 32P found inside
bacteria, 35S found outside bacteria
d. conclusion DNA is genetic material
6. Fraenkel-Carrat experiment 1957. Showed
that some viruses (tobacco mosaic viruses) use
RNA instead of DNA, i.e. instead of DNA being
transcribed and translated into protein, RNA is
original molecule from which DNA is made and
inserts itself into hosts cells DNA and becomes
part of it. Called retroviruses
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B. The structure of DNA 1. Discovery of a.
Miescher 1869, extracted white substance from
nucleus named muclein. Later called nucleic acid
because found to be acidic
b. Levene - 1920s i. A unite of DNA a
nucleotide one sugar, one phosphate and one
nitrogen base.
ii. bases are purines adenine, guanine, and
pyrimidines uracil, thymine
iii. DNA deoxyribose, double stranded,
thymine RNA ribose, single stranded, uracil
iv. his error thought base pairs were in equal
amounts
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c. Chargaff discovered complementary nature of
bases (see table 14.1, p. 285), i.e. Chargaffs
rules AT, CG, number of purines number of
pyrimidines.
d. Rosalind Franklin using X-ray
crystallography, determined DNA is a double
helix, 2 nm wide and helical turns every 3.4 nm.
e. Watson and Crick 1953, put it all together,
fig. 14.14, 14.15, p. 287 DNA is i. Double helix
with base pairs facing each other
ii. base pairs maintain 2 nm diameter and
therefore are purine pyrimidine
iii. base pairs are complementary and this is
based on hydrogen bonding.
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iv. sugars (pentoses) and phosphate (phosphate
functional group) are the sides of the molecule
(sugar phosphate form phosphodiester bond)
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v. The two strand are anti parallel, see trans
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C. Replication of DNA Prokaryotic 1. DNA
replication is semi-conservative, evidence
Meselson Stahl experiment.
a. three theories were proposed i. conservative
the entire double helix replicated at one time
ii. semi-conservative the 2 strands that make
up DNA pull apart at the h-bonds and 2 new,
complementary starnds form against each one
iii. dispersive DNA breaks into nucleotide
pieces and old and new re-form.
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b. The Meselson-Stahl experiment i. Based on a
very sensitive CsCl (cesium chloride) gradient
that can separate normal or light DNA, i.e. DNA
with regular nitrogen (14N) from isotopic or
heavy DNA, i.e. DNA with heavy nitrogen, (15N).
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2. DNA replication structures and molecules the
replication complex, see animations on web
site a. Requires precision and speed
b. Terms and molecules involved i. replication
origin where replication begins
ii. DNA polymerase III an enzyme that adds
nucleotides to the chain, see fig. 14.18, p. 290,
enzyme is a large dimer
iii. RNA polymerase (primase) is needed to
start the chain. Adds about 10 RNA nucleotides
to provide a place where DNA polymerase III can
start (bind). Later, RNA nucleotides are
replaced.
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iv. DNA replication always proceeds in the 5
prime to 3 prime direction
a. The strands pull apart. As soon as the 3 prime
end opens transcription of this strand begins.
This is a leading strand.
b. Once some of the molecule is pulled apart, the
other strand forms. This is the lagging strand.
(It must wait for the strands to keep opening)
c. Fragments (Okazaki fragments) are formed off
the lagging strand and later joined by DNA
ligase. Overall replication is
semi-discontinuous.
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3. The replication process a. DNA double helix
opens, initiator protein binds, helicases unwind
the strands, single strand binding protein
prevents rewinding, gyrases prevent torque.
b. 3 primer (RNA polymerase aka primase)
c. Complementary strands form (DNA polymerase I
removes primer and fills in gaps in okazaki
fragments
d. DNA ligase joins together the lagging strand
See animations on web site
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4. Eukaryotic DNA replication a. essentially the
same as prokaryotic DNA except chromosomes are
wound around histone proteins (i.e. are
nucleosomes) and chromosomal sections replicate.
(Called replication units aka replicas)
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b. Each replication unit has own origin of
replication therefore eukaryotic replication is
faster (happening simultaneously in many places)
c. How DNA unwinds (from nucleosomes for
transcription is not clearly understood.
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Chapter 15 Protein synthesis A. The Central
Dogma, see fig. 15.4, DNA to RNA to protein 1.
The big picture. see notes      
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B. The players molecules involved 1.
ribosomes aggregate of protein and RNA. Two
subunits, large and small, see fig. 15.2, p. 300,
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• 2. RNA (types)
• Ribosomal
• Messenger single strand, transcription
• c. Transfer single strand folded into
  cloverleaf shape, translation

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C. The genetic code read history p. 302-305 1.
Codon 3 nitrogen bases which codes for 1 amino
acid (aa)
2. breaking the code there are 4 nitrogen bases
and 20 different aa
a. 1 base codes for 1 aa four prime then there
would have to be 20 different bases
b. 2 bases code for 1 aa 4 to the second, only
provides 16 aa (we know there are 20)
c. 3 bases code for 1 aa 4 to the third 64.
This is the smallest number that accounts for the
20 aas. There are extras but some aas have
more than one codon and some codons are start and
end codons
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D. Protein synthesis - a two step process 1.
Transcription the players a. RNA polymerase
binds to the promoter (large protein complex with
5 subunits)
b. Only one of the two DNA strands is
transcribed -template DNA strand transcribed
(antisense strand)
-coding DNA strand is not transcribed (aka sense
() strand) i. promoter sites on DNA are about 60
base pairs long. Two six-base sequences are
found in most bacterial promoters (TTGACA,
TATAAA)
ii. In eukaryotes the TATAAA sequence is referred
to as the TATA box.
iii. Promoters vary in efficiency of
transcription
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2. Transcription the process (see website) a.
initiation RNA polymerase recognizes and binds
to the sequence of DNA called the promoter (in
eukaryotes other factors binding create a
transcription complex) DNA unwinds
b. elongation ATP (or GTP) breakdown provides
energy and starting molecule for chain to grow in
5 ? 3 direction as RNA nucleotides are added.
Areas containing RNA polymerase, DNA, and newly
forming mRNA are called transcription bubbles.
See fig. 15.7, p. 305. The bubble moves down DNA
about 50 nucleotides/second
c. Termination stop sequences form hairpin
loops that derails the molecule involved. RNA
polymerase, DNA and RNA dissociate
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d. Modifications after transcription -In
eukaryotes, 5 cap added to prevent breakdown of
mRNA
-also, 3 poly A tail protects 3 end from
breaking down during transport
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3. Translation the players a. tRNA (transfer
RNA)
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b. there are 45 different tRNAs. There are less
anticodons than codons (64) because of the
wobble, i.e. the first 2 bases are essential to
recognition, but sometimes the 3rd one can be
recognized by more than 1 tRNA molecule.
c. activation tRNA synthetase, see Fig. 15.11,
p. 307
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d. start and stop signals codons that do not
code for an amino acid aka nonsense codons UAA,
UAG, UGA stop codons, mark end of polypeptide.
AUG start codon, marks beginning of polypeptide,
initiates translation
4. Translation the process 3 steps. Review
cd rom and p. 308-309, there is a lot of detail
in book, focus on what is presented in notes. If
you get that, then you can get the fine points
from the book. Note -f-met tRNA (a tRNA carrying
a modified methionine) is always the first tRNA
to bind (a starter)
-binding may be assisted by proteins called
initiation factors a. step 1 initiation. The
small ribosomal subunit binds to mRNA.
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-tRNA with f-met attaches to mRNA at AUG (start
codon. anticodon on tRNA UAC
-the large ribosomal subunit binds and is
positioned so f-met tRNA is in the p-site.
-A second tRNA with its aa binds to the A site
-initiation complex is now formed
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b. step 2 elongation. The first 2 aa are joined
together (peptide bond) by the enzyme peptidyl
transferase
-the ribosome moves over one position to the
right. The tRNA that was in p-site leaves. The
tRNA in A site moves to p, called translocation.
A new tRNA with its aa moves into A site
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-the process repeats over and over until a long
chain of aas ( a polypeptide or protein) is
formed
NOTE if many ribosomes are moving along the same
mRNA molecule, its called a polysome. see fig.
15.10, -306
c. step 3 termination. The ribosome keeps
moving until it reaches a stop codon (UAG, UAA) -
Release factors (proteins) causes release of the
polypeptide chain and the initiation complex
breaks apart (including the large and small
ribosomal subunit) (next slide)
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5. Differences between protein synthesis in
eukaryotes and prokaryotes review p. 312. Main
difference editing of primary transcript in
eukaryotes,
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a. Prokaryotes entire DNA is transcribed and
translated
b. Eukaryotes entire DNA is transcribe and
spliced in nucleus with exons become secondary
transcript, leave nucleus, are expressed (make
proteins). Introns remaining in nucleus and not
translated
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