BIOL 311 Lecture Notes - Lecture 15: Wild Type, Zygosity, Covalent Bond

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Published on 6 Dec 2017
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Gene Regulation
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-Summary of the techniques seen so far
-PCR: amplifies DNA
-Dideoxy sequencing: sequences DNA
-RFLP: uses restriction enzymes to detect polymorphisms
-Northern blot: used to detect mutations affecting transcription
-Western blot: used to detect mutations affecting translation
-Southern blot: used to detect polymorphisms in DNA (focusses on the problems in replica-
tion)
-Mutations tell us what parts of the genome are important for what function
WHAT IS GENE REGULATION?
-Gene expression is when certain genes are expressed only at certain times or life stages
-Gene regulation is the regulation of the synthesis of a gene’s transcript and its protein product
-Gene regulation can be achieved by adjusting the transcription of DNA into RNA, or the
translation of mRNA into protein
-Stability of mRNA and post-translational modifications of proteins also play a role
-Gene regulation is a result of fluctuations or changes in the environment, for example, tem-
perature fluctuations and overcrowding
-Products of most genes are only required in certain cells at
certain times
-How does regulation occur?
-Molecular signals form outside or inside the cell lead to
the binding of regulatory proteins to specific DNA sites
adjacent to the protein-coding region
-Rate of protein binding modulates the rate of transcrip-
tion
-Transcriptional regulation in prokaryotes depends on genetic
switches
-Genetic switches are specific DNA sequences bound by
specific regulatory proteins under specific conditions
(NOTE: term “specific” used several times in this one sen-
tence indicates that the process does not occur at ran-
dom, it is highly specific)
-Operating the regulatory switches
-An activator (a regulatory protein) binds to the activator binding site (ABS — regulatory
DNA sequence) and assists RNA polymerase bind to the promoter
-The tighter the activator binds to the RNA polymerase, the more likely transcription will
happen
-Positive regulation means that the activator must be there for transcription to occur or to
occur at a higher rate
-Negative regulation means that the repressor must be absent for transcription to occur
-The repressor (a regulatory protein) acts as a road block, preventing the RNA polymerase
to bind to the operator (regulatory DNA sequence) and therefore read the sequence
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-How do the activators and repressors operate the switches in response to a cell’s needs?
-State 1: can bind DNA targets (ABS or operator)
-State 2: cannot bind DNA targets
-Two states are possible due to allostery
-Proteins can adapt to two different shapes, according to the state
-DNA binding region either binds ABS or the operator
-Effect of allosteric effectors on regulatory proteins
-Top half of the protein is the allosteric effect binding region, which causes a shape
change that allows the protein to bind to DNA via an allosteric effector
-Bottom half of the protein is the DNA binding region, which causes a shape change
which prevents the protein from binding to DNA via an allosteric effector
-Think of an allosteric effect as a molecular signal
-Lac Operon in E. coli
-Glucose is E. coli’s favourite carbon source because it is easy to work with in terms of mak-
ing ATP
-E. coli has to do more work to break down lactose because it is composed of two simple
sugars: glucose and fructose
-The lac operon
-The operon is the segment of DNA that encodes a multigenic mRNA + common promot-
er + regulatory region
-The multigenic mRNA is translated by the ribo-
some to form different proteins
-The operon consists of 3 genes: lac Z, lac Y,
and lac A
-lac Z: encodes B-galactosidase, which
breaks the covalent bonds holding the two
sugars (lactose and glucose) together
-lac Y: encodes permeate, which is a channel
that allows the entrance of lactose inside the
cell
-lac A: encodes for transacetylase
-Two switches regulating the lac operon:
-On/off switch
-The lac operon is only expressed in the
presence of lactose because when lac-
tose (allosteric effector) is present, it
prevents a repressor from binding to the
operator
-Lac repressor prevents expression of
the lac operon the lactose is absent
-Volume switch
-Controls the level of expression of the
operon — high when glucose is used by
E. coli
-The lac operon is only expressed at high
levels when lactose is present and glucose is absent
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