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Chapter 33: Structure of DNA and RNA
Supercoiling
Unwound DNA and supercoiled DNA are topological isomers = topoisomers
Most naturally occurring DNA molecules are negatively supercoiled (left-handed)


DNA is partly unwound and prepares DNA for processes requiring separation of DNA strands
such as replication or transcription



Positive supercoiling makes strand separation more difficult

Eukaryotic DNA is Associated with Specific Proteins
Eukaryotic DNA is tightly bound to histones = constitute half the mass of a eukaryotic chromosome
Chromatin = entire complex of a cell’s DNA and associated protein
Major histones: H2A, H2B, H3, H4
 2 of each histone come together to form an octamer


Eight histones in the core = DNA wraps around them



Each histone has an amino-terminal tail that extends out from the core structure
o

Flexible, positive charged tails



DNA backbone is relatively negatively charged, therefore strongly interacts with positively



charged histone tails
If histones are acetylated = open chromatin state and the gene will/can be expressed
o

Histone acetylation decreases the affinity of the histones for DNA, making additional
genes accessible for transcription

RNA Can Adopt Elaborate Structures
Stem-loops = when two complementary sequences within a single strand come together to form a
double-helical structure
o Usually made up of Watson-Crick base pairs
o Metal ions such as Mg2+ assist in the stabilization of these structures

Chapter 34: DNA Replication
DNA is Replicated by Polymerases
DNA polymerase = catalyze the step-by-step addition of deoxyribonucleotides to a DNA strand
DNA polymerases requires a primer to begin synthesis
Elongation of the DNA strand proceeds in the 5’  3’ direction
DNA polymerases have distinct nuclease activity that allows them to excise/correct any mistakes by a
separate reaction
DNA Helicase acts as a wedge to force the separation of the two strands of DNA

DNA Replication is Highly Coordinated
DNA is separated by DNA helicase
o Helicase requires ATP to separate the two strands of DNA
o DNA helicase is a hexomer (6 components)
o 2 components will have hydrolyzed ATP bound, 2 will have true ATP bound, and
2 will have nothing bound
o

Each time 2 ATPs are used, the helicase will move down the DNA two more
bases

o

2 ATPs = 2 bases that are getting separated

As helicase unwinds DNA, becomes supercoiled and positively charged
If it keeps supercoiling it will cause breaks to the DNA  need to find a way to fix this (Topoisomerases)
Topoisomerase = enzymes cut the DNA on purpose and allow DNA to become negatively coiled again
and it reconnects the DNA back together
o
o
o

Knicks DNA at one or both strands
Releases supercoiling
Connects back together

Topoisomerase I = will only knick 1 DNA strand
o

Allows DNA to squiggle around the phosphate bond and then seals the break

Topoisomerase II = cuts both strands
o
o
o

Introduces negative supercoils
PROKARYOTES = known as DNA gyrase
Ciprofloxacin = targets Topoisomerase II  stops it from reattaching the DNA back together

DNA Polymerase
o
o

Adds nucleotides to daughter strand of DNA
Depends on the existing 3’ hydroxyl

Polymerase called RNA polymerase forms a primer = does not require 3’ OH
Pol-alpha = initiating; binds right to where the primer is and will add about 20 bases with no
proofreading activity
Pol-epsilon = adds hundreds of base pairs to the leading strand
Pol-delta = adds hundreds of base pairs to the lagging strand
Pol-epsilon and Pol-delta have proof reading activity = have exonucleases
o Exonuclease = removes mismatched nucleotides from the 3’ end of DNA by hydrolysis

o

o 3’  5’ hydrolysis
If wrong nucleotide is added = malformed product is not help as tightly in the
polymerase site and is like to flop out and find itself in the exonuclease active site

How does this enzyme know whether a newly added base is correct?
1
...

2
...

After the addition of a new base, DNA is pulled by one base pair into the enzyme
...
This pause provides additional time for the strand to wander into exonuclease site
...
Take bacterial cells and expose them to potential antigen
...
Grow cells on media lacking a particular amino acid that the bacteria cannot make
...
If the reagent/compound is not a mutagen, cells will not be able to make the amino acid
and won’t be able to grow on the media
...
If the reagent/compound is a mutagen, it will be able to make its own amino acid that
the media is lacking and will grow
...
Possible for humans to infest something without mutagenic activity and then we create it into
something that has mutagenic properties in our bodies
...
Point mutations
a
...
Could be inconsequential because multiple codons can code for the same amino acid
2
...
Frameshifts
a
...
Deletions
5
...
DNA breaks
7
...
Chromosomal
a
...
Duplicate a whole chromosome = more expressions of genes than you’re supposed to
have
c
...
Translocation = parts of the chromosome are switched around
i
...
Mutation/damage needs to be recognized
2
...
Gap must be repaired with polymerase and ligase
Multiple types of repair systems:
o

Mismatch repair
1
...
Determine which base pair is incorrect

3
...

o

The parent strand of DNA will have the methylcytosine
...


In prokaryotes:
o

MutS attracts MutL  form a complex  recruits MutH

In eukaryotes:
o

MSH2/MSH6 atttracts MLH1/PMS1  complex  MutH

MutH = 5’  3’ exonuclease
In addition to heavy methylated sit, Okazaki fragments and PCNA fragments can be used to
recognize the nascent strand
o

Nucleotide excision repair

Repairs physical damage such as that from UV light, thymine dimers
1
...
Excision of a 12 nucleotide fragment (by UvrA) that surrounds the damage
3
...
DNA ligase connects the knick
UvrA in prokaryotes = XPC in euk
UvrB in prokaryotes = XPA and XPD in euk
UvrC in prokaryotes = ERCC1-XPF and XP6 in euk
In mammalians = 30 proteins involved in nucleotide excision repair
Two types of Nucleotide Excision Repair:
1
...
Transcription coupled NER = acts specifically on genes where RNA Pol II recognizes damage and
stalls
o

Base excision repair

Repairs alkylation to a base, oxidation of a base
Enzyme called DNA glycosylase = bound to DNA
1
...


2
...
Deoxyribose phosphodiesterase comes in and removes the backbone unit  whole nucleotide
missing
4
...

5
...

Break repair processes:
o

Non-homologous end joining

Used to fix double stranded breaks (DSB)
a
...
Takes about 30 minutes = quick
c
...
Ku recognizes breaks and binds to the cut ends
...
Nuclease will chop back the ends
...
Polymerase comes in and fills the gaps
...

Mutations are going to form because it’s chopping off some DNA before some of the break is fixed
...
Polymerase mu comes in and fixes
without a template
...
Takes about 7 hours
b
...
Requires homologous chromosome = diploid organism is fine
1
...

2
...
It wants some there to be able to use to add bases
...
Filled in by DNA polymerase
...
Unique structure known as the holiday structure
...

5
...
Ligation
...
”
Direct Repair
Mammals = one form of direct repair
Ex: Alkylation, or methylguanine

1
...

2
...

3
...

What happens if Repair Mechanisms are Drisrupted?
Cockayne Syndrome = mutations in transcriptional stalling recognition pathway, lack of detection of
DNA damage
XP = mutation in gene involved in nucleotide excision repair

Chapter 36: RNA Synthesis and Regulation in Bacteria
DNA  RNA is known as transcription
o

Catalyzed by RNA polymerase

RNA polymerase requires:
1
...
The sequence of the template strand of DNA is the complement of that of the RNA
transcript
...

2
...
A Divalent Metal Ion
a
...
Locate the transcriptional start site

2
...
RNA synthesis is initiated = no primer is required
4
...
Modify the RNA
6
...
DNA template
2
...
Divalent metal ion = magnesium or manganese
a
...


Eukaryotic:
Phosphorylation of RNA Pol II = change conformation = change from initiation to elongation
TFIIH = will stick around for elongation
Most other TFs will be released after initiation occur
Elongation factors suppress pausing by the polymerase
Termination
Prokaryotes:
Protein independent
a
...
Forms a loop structure
i
...
Weak interaction between
RNA and DNA will allow DNA to come away
...
Will form a structure that surrounds RNA and zooms around RNA and looks until it finds a
specific sequence that is 72 bp long
b
...
Stop transcript
...

Pausing during elongation at a poly-A sequence (could be U or U-G rich)
...
Eukaryotes Regarding Transcription
Eukaryotes: 3 different RNA polymerases
o

Pol 1 = ribosomal RNA synthesis

o

Pol 2 = micro and mRNA synthesis

o

Pol 3 = transfer RNA

RNA polymerase has a clamp domain that allows for processivity of domains

Fingers, thumb, and palm domains
Proofreading activity for RNA polymerase = not very picky though
On top of RNA polymerase there are other transcription factors that act as a complex and will interact
with RNA polymerase
A lot of processing of mRNA
Nuclear membrane = DNA in nucleus  DNA brought out of nucleus to a ribosome where translation
occurs
Prokaryotes:
DNA is accessible to different proteins
Ribosome is working in tandem with RNA polymerase
RNA polymerase is already a complex
Transcription Factors
o

Help RNA Pol II to bind to a promoter region  help it figure out what genes it’s supposed to be
transcribing

o

General TF complexes
o

TFIIA, TFIIB, TFIID, etc
...
It wraps around and
it interacts with it on the same piece of DNA
...

DPE element = bound by different transcription factors and regulating the expression of that gene
Regulation of RNA Pol II is complex = not just TATA box… promoter elements and enhancers play a role
RNA Pol I = 2 main sequences:
1
...
Ribonuclease initiator element
Most regulated because not every cell needs the genes it’s transcribing
RNA Pol III = very specific sequences that are downstream from the TSS… bind to A, B, C blocks
Nucleosome Position During Transcription
DNA wrapped around histone core = nucleosomes
At transcription bubble DNA is breaking away from the histone core so that the bubble can exist
Part of the DNA is always in contact with the histone
Termination of transcription: “The sequence signals for termination of transcription are contained
within the transcript itself
...
Because of this, prevents
transcriptional elongation, prevents bubble from occurring
Original therapy used to treat cancer

Chapter 37: Gene Expression in Eukaryotes
Problem of DNA Access
Epigenetics = modifications to the DNA or to the histone proteins that DNA is wrapped around that alter
the structure of the chromatin and that allows transcription factors to bind or not to bind
Reversible = go on and off = can use as a therapeutic agent
Cell needs to access genes for transcription
Chromatin Structure
Within nucleosome, 8 total histones… 2 of each 4 different histones
H2A, H2B, H3, H4
Tails come out of the histone proteins
C-terminal tails = can be modified by chemicals
AC = acetylation
PH = phosphorylation
ME = methylation
Writers, Erasers, and Readers

Enzymes puts on modifications = writer
Remove modifications = eraser
Modifications serve a purpose, something has to read them to cause a downstream effect = reader
Histone Acetylation and Transcription
Acetylation can be turned on and off
DNA backbone is negatively charged, so it interacts with positive charge found on the amino acids on
histone tails
...

Enzyme HAT = pushes reaction forward = will acetylate histone tails
Enzyme HDAC = reverses reaction = push reaction back and remove acetyl groups
If chromatin is acetylated, separation of DNA and histones = transcription can occur, gene can be
expressed
Closed chromatin state = no access for TFs
DNA Methylation
Modifications on DNA level
Cytosines can be methylated in DNA
Important because of mismatch repair
Happens at 5’ carbon position of cytosine
3 enzymes: DNA methyl transferases
DNA that has no methylation
DNA methyl transferases can methylate DNA at the cytosines
During DNA replication, parent strand now has methylation
Methylation of cytosine does not disrupt base pairing
Methyl binding DNA proten (MBD) = recognizes and binds to the methylated Cytosines
-CANNOT transcribe gene if this promoter is methylated
If promoter is not methylated = TFs can come in, recruit RNA polymerase, and transcribe
Methylation is inhibition of transcription
Methylation of DNA = gene will be silenced
Acetylation of histone tails = open chromatin state, gene expressed

Nuclear Receptor Regulation of Expression
TFs play an important role
Estradiol comes in and binds to nucleoreceptor = changes conformation of protein = dimerization =
recruitment of co-activators
Co-activators such as HAT can be recruited to add even more acetylation to chromatin to make it more
accessible
-coactivators work with other proteins to form large complexes that modify chromatin and the
transcription machinery to regulate gene expression
Drugs that mimic hormones and toxins can activate or repress receptors

Chapter 38: RNA Processing in Eukaryotes
Processing of Eukaryotic rRNA
RNA Polymerase I transcription results in a single precursor (45S in mammals) that encodes three RNA
components of the ribosome:
18S rRNA
28S rRNA
5
...
8S rRNA are the components of the large ribosomal subunit (60S)
Nucleotide modification = methyl groups are added to nucleotides, conversion of uridine to
pseudouradine
...
Leader and trailer sequences are cleaved off
2
...
Modification of some nucleotides
a
...

4
...
Polymerase comes in and adds amino acid attachment site

b
...
First phosphate will be hydrolyzed and removed
2
...
First phosphate of GTP will attack diphosphate at 5’ end
b
...
Opposite direction
3
...
If only methylation, called the 0 cap
b
...
First base methylated = 1 cap
ii
...
N means that it could be any base
...

Branch site = adenosine always there…
...
A in the middle of the intron
a
...
Frees up an end = forms a lariate structure = phosphate group is now attached
to that adenosine
2
...
Will attack the phosphate at the other splice site
b
...
There are enzymes that come in and mutate the transcript to form a different
codon
...
CAA would be a codon and can be deaminated to UAA, which will now be a
stop codon
...

If there is something funny about a transcript or gene wants to turn expression off, can degrade the
RNA, cleave off what protects it and then chop it up
...
”

Chapter 39: The Genetic Code
Transcription vs
...
Three nucleotides encode an amino acid
2
...
Code has no punctuation
4
...
Code is degenerate
6
...
”
Central Dogma
DNA to RNA to protein
AUG = start codon = where protein being translated will begin
Reading Frame
If it’s shifted  proteins are completely different
AUG is important  tells ribosome where the reading frame is
If you have a single base insertion, it would shift reading frame completely and can cause mutations

Chapter 40: The Mechanism of Protein Synthesis
tRNA Aminoacetylation
Amino acid attachment site will be added to the 3’ end of the tRNA
Will always be ACC if reading 5’  3’
Flexible = important
Sometimes there is an “extra arm” that is variable in length
The Link Between Transcription and Translation
Anticodon = on tRNA that matches with codon sequence for mRNA
Wobble when it comes to codons = third base tends to be variable between different amino acids
Wobble in codon/anticodon pairing, too = 1s t and 2nd base usually match perfectly, 3rd base don’t care as
much
“Charged” tRNAS
Needs to be charged = getting energy in the form of ATP
Amino acid becomes adenylated
Add tRNA onto charged amino acid
AMP is lost
Esterification reaction that connects amino acid to tRNA at that CCA sequence at the end of tRNA
AARS
Specificity of activation site
Proofreading activity
Synthetase = activating site and editing site
If amino acid is added to tRNA and doesn’t fit in the editing site it will be removed and proper amino
acid will be added
If amino acid is added and it does fit into the activated site, AARS says it’s okay, we can release the
whole tRNA
“Which of the following best characterizes the relationship between amino acids and tRNAs? The
activation of an amino acid by formation of an aminoacetyl-tRNA is coupled to the hydrolysis of ATP to
AMP + 2Pi
...
methionine
Eukaryotes:
Initiation factors bring in Met-tRNA
Bring to 40S subunit
Will slide along until it finds first AUG
Hydrolysis of ATP
Methionine will be brought in by IF = binds to AUG
As it slides along, utilizes GTP
Each codon that it slides along it uses another ATP
Once AUG found = 60S subunit comes in
More Ifs in eukaryotes
Main IF will bring in Met-tRNA = binds to 5’ mRNA
Other Ifs that bring to PolyA tail and make a circular mRNA = promotes the process to go continuously
Elongation
Elongation factors (Ef-tu)
Ribosomal complex
Polypeptide in tRNA P site

Ef-tu will bring in aminoacetylated tRNA into A site
If everything matches up, Ef-tu will leave aminoacetylated tRNA there
GTP will be hydrolyzed
Polypeptide chain that’s already there will be added on to new amino acid brought in
Polypeptide chain added to tRNA in A site and everything shifts over
Transpeptidation
P site has tRNA that contains the polypeptide
A site = new tRNA that has the proper amino acid that should be added next to the polypeptide chain
No free energy is required
Moving the Ribosome
EF-G (bound to GTP) = uses GTP to shift over the ribosome through hydrolysis of GTP
Move tRNAs through conformational change of ribosome
Termination
Stop codon = no tRNA that matches it
If no tRNA comes in, releasing factor can come in because no new amino acid comes in to be transferred
Through the use of water, releases polypeptide chain
“How many nucleotide triphosphates are hydrolyzed per amino acid incorporated into a growing
polypeptide chain during protein synthesis on the ribosome? 2 GTP”
1 used to move ribosome, 1 used to bring in tRNA
Eukaryotic Ribosome Components
Slight difference between euk and pro


Processes are the same



Most differences are nomenclature

Eukaryotic = 40S and 60S
Prokaryotic = 50S and 30S
Small e in front of proteins means eukaryotic
Peptide transfer to water instead of amino acid so that it can be released
“Which of the following best characterizes the termination of translation? Release factors are proteins
that catalyze the transfer of the polypeptide from tRNA to water
...
Ribosomes = larger in euks
2
...
Initiation = first AUG in mRNA
a
...
eIFs = many more in euks
c
...
Eukaryotic mRNA is circular
5
...
Homologous proteins EF-tu and EF-G are EF1a and EF2
6
...
Other proteins can interact with it = chaperone proteins = can move the protein where it’s
supposed to go
a
...

b
...
Some signal as part of the protein that tells it where to go
d
...
Glycosylation = common, usually occurs right at ER
a
...
Usually extracellular/membrane proteins
2
...
If a protein doesn’t look right or something wrong, or cell doesn’t need it, cell will
ubiquitinate it
...
Methylation, acetylation of histones
Protein Synthesis as Drug Target
Ribosomal subunits are different in euks and proks
Inhibition of Translation Can be Deadly
Cleaving adenine affects the function of rRNA  results in inhibiting the binding of elongation factors
Translation Disruption in Disease
Vanishing White Matter disease
Mutations in eIF2
Nerve cells break down = vanishing neurons, “holes” in the brain

Biotechnology and RNAi
Biotechnology: living things to create products or to do tasks for human beings
Can use plants, animals, micro-organisms, and biological processes to our benefit
Used in many industries, including agriculture, pharmaceutical, biomedical, environmental
Biotechnology, Then and Now
Biotechnology as existed for centuries:
1
...
Animal breeding to obtain useful animals
3
...
Transgenic technology
2
...
can be inserted into a different species
3
...
This manipulation goes far beyond the changes that occur naturally during evolution or changes
due to selective breeding
5
...
Something that you're exposed to that alters the chemistry or a process in the body and has some
effect or outcome
DNA plasmid
Double stranded RNA
Protein (insulin)
Small molecule drugs
^^all could be potential therapeutics because they all can alter something in the body
Types of therapies we are using are more related to biotechnology now more than they ever used to be
Making Drugs out of Proteins
Transcription and translation are very complex processes – cells have developed good mechanisms for
these
Easier to use micoorganisms to make the proteins for us
Easier to make a protein to use as therapy for a microorganism to make it for us
Restriction Enzymes
Restriction enzyme = another term for restriction endonuclease
Cuts straight down the middle = blunt ends (rare)
Some leave overhangs = sticky ends (preferable)
EcoRI = name tells you where it comes from
Comes from E
...
Good for cloning two different
pieces
...
Coli plasmid
Make a piece of DNA that contains our gene of interest – whatever gene we want to make protein for
Copy it from the genome by PCR and add sequences at the end that when they are cleaved by the RE,
will be complementary to the DNA on the plasmid
Sticky ends = 2 pieces of DNA will come together, ligase comes in to fix knicks
DNA is part bacterial plasmid and part of whatever gene we wanted in there
Recombinant DNA
Take that plasmid, put it back in bacterial cell and let it grow
Plasmid also grows so we can make more plasmid that way, purify DNA, and use it how we want to
We can put a promoter in the plasmid that will be active in the bacteria, now the bacteria will make the
human protein
Sometimes sticky ends will ligate back with itself… Religation of plasmid and gene to themselves
Translation similar in eukaryotes and prokaryotes
Screening for Insert*
Antibiotic resistance
Only those bacteria that have the plasmid with the correct insert in it will grow
Only cells that contain the plasmid will be able to grow/divide and form colonies on the antibiotic agar
*Gene will only be expressed properly if insert is in there if there is antibiotic resistance* ??
Color = instead of having antibiotic resistance, has Lac Z
It’s not expressed if insert is in there
Not expressed if insert isn’t there
All bacteria will grow, but only the ones without the insert will make a blue color
Blue color = insert isn’t there

White color = insert is in the middle of the Lac Z gene, just isn’t being expressed
The Story of Insulin
Early 1900s = patients ingested animal pancreas
Insulin isn’t orally available = broken down in GI tract = won’t get to receptors
1922 = injects dog insulin into Type I diabetes patient
It did help = but not ideal because it could cause an immune reaction
Not really as purified as much as it should have been or could have been
1978 = synthetically manufactured human insulin
Used bacteria = cloned human insulin gene into bacterial plasmid = took it out, purified it
1980s to current = availability of human insulin revolutionized the treatment of diabetes
How Exactly did They do This?
Human pancreas tissue or any human tissue = insulin is in every cell of the body
Can’t use genomic sequence to clone into bacteria = NEED TO USE RNA
1
...
Need to make cDNA
3
...
Transform this into bacteria
5
...
Bacteria will make that protein and it can be purified
Reverse transcriptase = makes complementary DNA from RNA template = yields cDNA
1
...
Deoxynucleotides
RNA isn’t very stable  why it can’t be cloned
Has to be double stranded, cut with RE, must have a sticky end
cDNA Synthesis and PCR
All mRNA has a poly-A tail
Utilize this to our benefit… reverse transcriptase needs a primer
Put in an oligoT primer = binds to poly-A tail
Reverse transcriptase will start reverse transcribing the mRNA
Enzyme goes backwards on mRNA and adds complementary nucleotides to original primer that’s there
One strand is RNA… one strand is DNA  separate the two strands and use as template for PCR

“Which of the following reactions is catalyzed by reverse transcriptase? 5’  3’ polymerization of DNA
from an RNA template
...
Denature DNA with heat
2
...
Allow primers to anneal to DNA template
...
Primers are important
b
...
20 base pairs long - anneal to end of sequence that you want to amplify
3
...
72 degrees
b
...
Bacteria thermos aquaticus = use this polymerase
Quantification of PCR Product
Why would we want to do this?
cDNA is template: represents how much transcription is occurring
Situation where you might be interested in finding amount of transcript? Tumor
...
Which of the following
best describes the mechanism by which 2’, 3’-dideoxyribonucleotides cause chain termination in these
reactions? 2’, 3’-dideoxyribonucleotides can be incorporated into DNA but prevent subsequence chain
elongation

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