BIOSC 0160 Study Guide - Midterm Guide: Deoxyribonuclease, Dna Supercoil, Leucine Zipper
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Study Guide Exam 2: Epigenetics: inheritance patterns not explained by sequence of DNA itself.
1. What is the purpose of restriction-methylation systems in bacteria?
● Restriction enzymes are the prokaryotic version of an immune system, they cut out foreign DNA.
They break the sugar phosphate backbone to digest DNA that is not methylated. Each restriction
enzyme cuts a unique sequence so there are thousands of different restriction enzymes. Methylase
adds methyl groups to self DNA to prevent it from getting cut.
● If cut straight thought double stranded backbone=blunt ends, if stagger cuts one side, then the
other a few bps down=sticky ends.
2.Each restriction endonuclease cuts at a unique sequence of DNA called a restriction site. Yet many of
these different restriction sites have one thing in common. What is a common characteristic of restriction
sites?
● They are palindromic. Ex: 5’-GAATTC-3’ in Ecoli. EcoRI cut non methylated G-A strand.
● 1/4096 chance of finding that sequence b/c ¼ six times.
3. What are sticky ends? Why are compatible sticky ends important in cloning?
● 5’ overhang ends of palindromic sequences cut by two separate restriction enzymes (with
endonuclease activity) near each other that reanneal to other fragments of complementary DNA
(usually the transgene being inserted) and get sealed together by ligase. (for the purposes of
cloning use two different restriction enzymes so the vector only matches the way you want it to
face). Theoretically could use one restriction enzyme and it will just cut where ever that sequence
is.
4. A restriction enzyme has a single restriction site in a plasmid. If a digest is performed, what technique
would be used to determine if the plasmid had been cut?
● Gel electrophoresis: different sections will weigh different because portions have been cut out
(DNA runs to the red because of overall charge of phosphate backbone).
● (Short Tandem Repeats: STRs, know that its the same sequence 4-11 bps (or some variation with
crossover during meiosis)in everyone just in different numbers of repeats and therefore varying
lengths, used with PCR for genetic fingerprinting).
● Single Nucleotide Polymorphisms: SNPs: can be good as marker for where diseased chromosome
is, detected with restriction enzyme if mutation lies within restriction site
5.What features must a plasmid possess to make it a good cloning vehicle?
● Ori of replication so get recognized by replication machinery of host
● Promoter
● Cloning sites with unique Restriction sites
● One or reporter genes such as selectable marker genes with easily visible expression (later help
recognize which host cells have taken up the gene). Antibiotic resistance gene
● Signal for transcription termination
● Sequence necessary for binding of bacterial ribosomes to mRNA (also must be inserted into
replicon: the part of the host cell that contains ori: can be spliced into there directly or put into
vector to be taken up)
6.What are two difference types of selection used in cloning a foreign piece of DNA into a plasmid in
E.coli? (Note: In prokaryotes=transformation, in euks=transfection)
● Antibiotic resistance: those that took up the transDNA will grow on antibiotic plate
● Visual Selections:
○ GFP: Green fluorescent protein: glows under light if uptakes this insert
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○ Blue white selection: recombinant DNA had lacZ cut out and replaced with target DNA
so should have function of B-galactosidase. Cells grown in X-gal (substrate for B-
galactosidase), mimics lactose but isn’t used by the cell, instead cleavage causes it to turn
blue and accumulate inside the cell so the cells that turn blue still have lacZ, the white
colonies are the ones that took up the recombinant DNA and don’t have lacZ.
● (screening: take all the white cells, grow on another plate, have a complementary probe for you’re
for, because the sequence could be there but backwards or flipped, so want the forwards one, so
probe picks out the exact colony you want with the right sequence)
● Then modify that gene with things to make it compatible to a mammalian cell: add promoter and
transcriptions factors etc.
7. Consider regulation of transcription in prokaryotes.
a. How many types of RNA polymerases are found in a prokaryotic cell?
● One, binds at -10 to -35 bps upstream of transcription start site.
b. What is a sigma factor?
● Proteins in prokaryotic cells that bind to RNA polymerase and direct it to specific classes of
promoters to initiate transcription. Sigma 70 is most common.
c. Why do bacteria have multiple different sigma factors?
● Under stress conditions, different sigma factors bind to different promoter sequences to transcribe
and express different genes to survive in conditions.
● Sigma 70 is constitutively expressed, others are not.
d. Where are operators located relative to a bacterial promoter?
● A region between promoter and first gene. Functions as an on-off switch for transcription.
Repressor protein binds to the operator and blocks RNA polymerase from moving forward.
e. What binds to an operator?
● Repressor proteins (co-repressor and inducer binds to these)
● Activator proteins (co-activator/inducers binds to these)
f. Where are activator-binding sites located relative to a bacterial promoter?
● Directly before operon promoter (after repressor gene promoter)
● Transcription factors bind to ABS to allow transcription.
g. What activator and coactivator control the optimal expression of genes regulated by catabolite
repression?
● Activator: cAMP Receptor Protein
● Co-activator: cAMP
h. Explain the difference in gene regulation between a repressor-inducer system of gene regulation and a
repressor-corepressor system.
● Bind at repressor binding site on operator, blocks RNA polymerase from moving forward from
promoter to genes. Generally bind to regions of DYAD symmetry. Important because
transcriptional factors generally bind to dimers, so makes sense there would be matching parallel
binding sites. Inducers bind to repressors, induces transcription
● Prokaryotes: Default is to have genes ON (but does depend on envt signals a little for how
strongly they’re turned on). Inducible systems: controls catabolic pathways: when genes only
want to be expressed in presence of plentiful reactant. turned on only when substrate is available,
negative feedback regulation. Default is to be on. If breaking something down, its negative
regulation, the more allolactose there is, it induces repressor, so can induce transcription of lac
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gene that will break allolactose down. have other trans acting inducers that inhibit repressor and
induce transcription.
○ Allolactose induces the lac operon by binding to repressor. N
● Prokaryotes: transcription initiated when RNA polymerase binds to promoter. Regulates in
operons (promoter, operator, gene cistron). Trans units bind on operator. Repressible systems:
when repressor’s default is to be off, a co-repressor binds to it, makes it bind to the operator so
inhibit repressor, induce transcription of enzymes to break down lactose.
○ Ex: Trp operon: when tryptophan is present, cell wants to stop making it, so tryptophan
acts as a corepressor to make the repressor bind to the operator and inhibit transcription.
Usually anabolic pathways. These default off because don’t need to waste E building
things unless prompted. Sometimes corepressor slips, still get some tryptophan
synthesized but not as much as before. Negative feedback regulation, the more
tryptophan there is the less it wants to make it so corepressor activates repressor to stop
transcription.
○ Positive feedback regulation: activator binding site activator, also needs
inducer/coactivator/. cAMP is an inducer for CRP activator.
● Eukaryotes: Transcription initiates when many proteins including RNA polymerase binds to
promoter. general transcription factors act as euk promoters. Specific transcription factors must
bind to the target promoter along with GTFs before the RNA Polymerase II can bind.
● Enhancers: cis acting elements: specific regulatory sequences, upstream, downstream or in the
gene. TFs bind to them to increase rate of transcription. Activator binds to them (or to promoters)
and inducers bind to activators
○ When enhancer far away from promoter, activator proteins bind and interact with GTFs,
each activator increases rate a little bit more.
○ Sometimes the activator proteins bind to mediator protein complex, a multiple protein
complex that also interacts with many other proteins, transcription factors, activators, etc.
Mediator relays signals from TFs directly to polymerase II TIC, facilitating TF dependent
regulation of gene expression.
○ If within 200 bps=promoter proximal elements, if further than 200 bps=enhancer
○ When activator binds to promoters: inducers bind to activator
○ When activators bind to enhancers, inducers also used but makes DNA loop in a certain
way to force interaction between TFs and certain DNA and polymerase.
● Silencers: cis acting elements: cannot be located in the gene (only upstream or downstream).
Could the promoter sequence so tightly that GTFs can’t get to it or blocks the promoter by sitting
on it. Usually found within 100 base pairs of transcription sites, but some can be far away. Also
need inducers.
i. What type of regulation would you expect for genes involved in amino acid synthesis in bacteria?
● repressor/corepressor system: Trp operon
8. Consider regulation of transcription in eukaryotes.
a. How many RNA polymerases are active in eukaryotic cells?
● Three
b. Which one is responsible for transcribing mRNAs?
● RNA polymerase II: transcribes mRNA, snRNA,
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Document Summary
Restriction enzymes are the prokaryotic version of an immune system, they cut out foreign dna. They break the sugar phosphate backbone to digest dna that is not methylated. Each restriction enzyme cuts a unique sequence so there are thousands of different restriction enzymes. Methylase adds methyl groups to self dna to prevent it from getting cut. If cut straight thought double stranded backbone=blunt ends, if stagger cuts one side, then the other a few bps down=sticky ends. 2. each restriction endonuclease cuts at a unique sequence of dna called a restriction site. Yet many of these different restriction sites have one thing in common. Theoretically could use one restriction enzyme and it will just cut where ever that sequence is: a restriction enzyme has a single restriction site in a plasmid. Single nucleotide polymorphisms: snps: can be good as marker for where diseased chromosome is, detected with restriction enzyme if mutation lies within restriction site.
