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FUNdamentals 1
09/17/08 (Dr. Ryan)
11:00 – 12:00
Slide 15 – General Organization of Operons
 Genes can be coordinately regulated by having them under the control of a single
promoter organized in operons.
o Picture here is in your book showing 3 structural genes
o Has an upstream promoter labeled “P”
o Has an operator sequence ladled “O” that controls the regulation of this
operator by interacting with proteins and can increase or decrease
transcription via the promoter
 These operons can be upstream, downstream or overlap with the promoter.
 The regulatory proteins bind the operator, which controls the expression of the
entire operon.
Slide 16 - Induction and Repression
 Increased in expression of the operon in response to metabolites is ‘induction’ and
the metabolites are called co-inducers
 Decreased synthesis in response to a metabolite is termed ‘repression’ and the
metabolite a co-repressor
 Some substrates induce enzyme synthesis/expression even though the enzymes
can’t metabolize the substrate
o these examples are ‘gratuitous inducers’ (such as IPTG in lactose operon)
Slide 17 - lac Operon Both Positive and Negative Regulation
 Most talked about of E. coli operons
 Examples of positive and negative regulation
 + 1 = start site of transcription w/ polysystronic messenger RNA that is produced
 Has -10 & -35 consensus sequences for sigma-factor recognition and RNA
polymerase binding at the promoter
 Also has a binding site for another transcription factor upstream of the promoter
 Also has an operator sequence which overlaps with polymerase binding site
where a repressor can bind
 Can have negative regulation (binding of this repressor), but can also have
positive regulation in times of glucose starvation.
 Multiple control mechanisms are typically the norm
Slide 18- lac Operon of E. coli
 Picture from book
 It shows the lac operon of E. coli
 3 structural genes
o lacZ – codes for Beta-Galactosidase
o lacY – codes for a membrane protein called Permease
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o lacA – codes for Transacetylase
Note the promoter with overlapping operator
Upstream of this promoter in the lac operon is a second promoter, which
expresses the lacI gene, which is the repressor of the lac operon
Slide 19 - The lac Operon Negative regulation
 The lac operon is negatively regulated
 The structural genes of the lac operon are controlled by negative regulation
 lacI gene product is the lac repressor
 lacI mutants express the genes needed for lactose metabolism constitutively
o In other words, if you have a mutation in the gene for the lacI gene
product that knocks out repressor binding, you can express the genes of
the lac operon constitutively
 The lac operator has palindromic DNA, where you have multimers of
transcription factors binding
 In this case, the lac repressor forms a tetramer
o DNA binding in the N-terminal domain
o Inducer binding at the C-terminal domain
Slide 20 - lac Operon lacI gene encodes a repressor
 Picture is in book
 The lacI gene product produces a short messenger RNA that encodes for a
repressor monomer. Four of the monomers come together to form a tetramer. That
tetramer binds to the operator blocking entry of the RNA polymerase and
transcription of the downstream genes: lacY, lacZ and lacA
Slide 21 - lac Operon Negative Regulation
 Operon is generally “off”; only fully “on” when lactose is present and glucose is
absent
o Fully on in absence of glucose & presence of lactose
 When no lactose is present: repressor is bound; inhibiting transcription of the
genes
 When lactose is present: lactose is converted to allolactose, binds to repressor,
causing it to fall off the operator and allowing transcription of the downstream
genes. These downstream genes are only maximally transcribed when glucose is
absent
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Slide 22 - Gratuitous Inducers
 One of the substrates of the lac operon is anything with the -galactoside linkage.
 -galactosidase is the first enzyme produced in the lac operon and it will cleave
this -galactoside bond here.
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Lactose is a substrate, but Isopropyl Beta-D-thiogalactoside (IPTG) is not a
substrate because of the thioester linkage that cannot be cleaved by galactosidase.
o However, it can bind the repressor and pull it off the operator therefore
turning on the operon (inducing the operon). Even though it is not a
substrate it can induce transcription of the operon.
A real substrate like lactose would bind, pull the repressor off, you get expression
of -galactosidase, the -galactosidase would cleave the substrate so it can no
longer bind to the repressor and the repressor would go back and shut off the
operon.
Once you’ve utilized your substrate, you do not want to keep making these genes,
so you shut it down. If you throw IPTG in there it will keep expressing because it
is not cleavable by -galactosidase
Slide 23 - -galactosidase (LacZ)
 -galactosidase, which is the first gene product of the operon, cleaves lactose to
glucose and galactose.
 The other enzymes in the lac operon are lactose permease, which enables the
import of lactose into the cell & acetylase, whose function is unclear.
Slide 24 - lac Operator Sequence
 The lac operator sequence shows dyad (paired complements) symmetry
 It has inverted sequences that look the same going 5’  3’ on the top strand of the
bottom strand.
 The operator site is palindromic and lies just downstream of the transcription
initiation site
 When the lac repressor (tetrameric) binds to the operator it blocks transcription
elongation by RNA polymerase
Slide 25 - Induction of the lac Operon
 To induce the lac operon: if you have any molecules around, any -galactosidase
that can bind to the lac repressor, it will induce a cooperative allosteric change
causing the repressor to fall off the operator and RNA polymerase can continue
elongation of the polysystronic message, and then you can synthesize the three
proteins.
 Again, once you make this -galactosidase, it can come and cleave the galactoside. Once you run out of lactose, then it will no longer bind to the
repressor and the repressor will bind to the operator and shut down the operon.
Slide 26 - lac Operon: Catabolite Repression
 The lac operon is also controlled by catabolite repression
 E. coli can use several sugars as carbon sources but prefers glucose because it
requires no energy to take it up
o If you have glucose around, the cell is happy metabolizing glucose, it
won’t turn on the lac operon
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The lac operon is an example of a glucose-sensitive operon (their expression is
reduced in the presence of glucose)
Catabolism of glucose inhibits expression of these operons – called catabolite
repression
This mechanism allows for the lac genes to be only partially induced in the
presence of lactose and glucose
Slide 27 - Catabolite Activator Protein - Positive Control of the lac Operon
 Why does the cell do this?
o Because there are ways to activate transcription in the absence of glucose.
This is via transcription factor Catabolite Activator Protein (CAP); also
called cyclic AMP receptor protein (CRP)
 This is an accessory protein to speed/activate transcription
 CAP binds as a homodimer
o N-term binds cAMP; C-term binds DNA
 Binding of CAP-(cAMP)2 to DNA assists formation of closed promoter complex
and can activate transcription
Slide 28 - Catabolite Activator Protein - Mechanism of Activation
 How does this work?
 Normally you have inactive CAP, but under conditions of glucose starvation
adenylyl cyclase will increase cyclic AMP levels.
 Low glucose = increased cyclic AMP
 Cyclic AMP can bind inactive CAP forming active CAP, this will bind to the
binding site that is upstream of the lac operon and increase transcription.
Slide 29 - Catabolite Activator Protein
 CAP protein in green
 Cyclic AMP will bind DNA upon binding
Slide 30 - CAP Activation of RNA Polymerase
 Here is the lac operon
 Note the RNA polymerase binding to the promoter
 Note the alpha-subunit of RNA polymerase
 When the CAP-cyclic AMP dimer binds it can interact with the C-terminal
domain of the alpha-subunit and increase transcription of the operon
Slide 31 – Dual control of the lactose operon
 This is a schematic showing the four conditions you can have in the presence or
absence of glucose and lactose
 + Glucose, + Lactose
o In the presence of lactose it can bind to the repressor and pull it off the
operator, but the operon is still off because you have glucose, so CAP isn’t
activated
 + Glucose, - Lactose
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o No lactose will cause the lac repressor to bind to the operator and shut
down the operon.
- Glucose, - Lactose
o CAP activated (cyclic AMP will bind CAP and bind the upstream
sequence), but no transcription because you have no lactose and repressor
is still bound
- Glucose, + Lactose
o In the presence of lactose, you have the receptor coming off the operator.
Without glucose you have cyclic AMP binding CAP and now you can
increase and get full expression of the operon.
Slide 32 - Regulation of the Arabinose (araBAD) Operon
 Again, the lac operon shows negative regulation via the repressor and positive
regulation in catabolite repression in activation of CAP
Slide 33 - The trp Operon - Co-repressor Mediated Negative Control
 Second operon in discussion.
 This operon has 5 genes, which encode 3 biosynthetic enzymes for the production
of the amino acid tryptophan.
 The 5 structural genes are located in a single operon with a single promoter
upstream and they are preceded by a short region called the trpL, which is a leader
region that encodes a leader peptide. This is important for the regulation of the trp
operon.
 There is another site of the chromosome that contains the trpR gene, which
encodes the repressor.
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The trp operon is a co-repressor mediated negative control
Encodes a leader sequence and 5 enzymes for tryptophan biosynthesis
trp operon is always “on” unless tryptophan levels are sufficiently high to turn
“off”
o If you have plenty of tryptophan you don’t need to make these enzymes
and you shut it down
o If you don’t have a lot of tryptophan, then you want expression of these
genes.
Tryptophan is the co-repressor
Trp repressor binding excludes RNA polymerase from the promoter
Trp repressor also regulates trpR and aroH operons and is itself encoded by the
trpR operon. This is autogenous regulation (autoregulation) - regulation of gene
expression by the product of the gene.
Slide 34 - trp Operon
 How does this all work?
 Here you have the trpR gene encoding the messenger RNA for the repressor
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In the presence of plenty of tryptophan, the tryptophan is the co-repressor, it binds
the inactive trp repressor forming an active repressor complex that then binds to
the operator and blocks expression at the promoter of the trp structural genes.
Slide 35 - Regulation of the trp Operon by Repression and Attenuation
 The trp operon also has a second method of regulation where it senses the level of
tryptophan after transcription at the promoter has already begun. It does this in the
trp leader region.
 When tryptophan levels are high, you get an attenuated transcript (the transcript is
terminated). There is an intrinsic termination sequence that is formed and
transcription stops.
 When you have low tryptophan levels, causes stalling in the leader sequence with
ribosome stalling the formation of different secondary structures in this leader
region in the transcript and you get expression of the trp messenger RNA.
 This is called transcription attenuation. If the transcript has already started, but
while the RNA polymerase is transcribing through the operon and you have
translational ribosomes binding the nascent transcript and forming this leader
peptide, you can sense tryptophan levels because of a couple of tryptophan
codons in this leader region
 If tryptophan levels are high, you have plenty of charged triphenyl tRNA. The
ribosome proceeds to that leader region quickly and blocks the formation of many
secondary structures, which causes the early termination of the transcript.
Slide 36 - Attenuation
 Affects transcription by adjusting the rate at which transcripts are completed after
they are initiated.
 When particular mRNA secondary structures form, transcription is likely to
terminate. The rate at which ribosomes progress behind the polymerase will alter
the RNA structures that can form. Thus, the transcription of enzymes for
synthesis of an amino acid can be adjusted to the rate at which the codons for the
amino acid are translated.
 The trp operon: regulated by a Repressor and by Translational attenuation
Slide 37 - Attenuation
 The need for tryptophan is tested during translation of the “leader” sequence
(“trpL”) on all mRNA initiated at promoter.
 trp expression is modulated by the tRNA-tryptophan (“charged tRNA”) available
to translate codon UGG. It has a couple of these tryptophan codons in the leader
region. If the ribosomes translate these codons rapidly, transcription will stop
because:
o Different segments in the leader mRNA sequence can pair with each other
to form alternative stem-loop structures.
 If ribosomes “stall” on the trp codons, the RNA structure formed is
not a terminator.
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 TRANSCRIPTION OF trp OPERON PROCEEDS
If ribosomes translate the leader and pass the trp codons quickly,
an intrinsic terminator is formed.
 TRANSCRIPTION OF trp OPERON TERMINATES
Slide 38 - The mechanism of attenuation
 Note the leader sequence
 Note the structural genes to the right side
 When tryptophan levels are low, these two red boxes here are the tryptophan
codons in the leader peptide
 If the tryptophan levels are low, the ribosome stalls here on the leader peptide at
the tryptophan codons, allowing the formation of the 2,3 antiterminator hairpin.
 In the 5’ end of this transcript there are four stems that can form three different
stem loops.
o 1,2; 3,4 or 2,3.
o If the 2,3 is formed, it is the antiterminator and you will get expression of
the downstream genes.
o It blocks the binding of 2 to 1, so 2 pairs with 3 and the transcript
proceeds because you want to make these enzymes because you want to
make more tryptophan
 When tryptophan levels are high, the ribosome blows through the leader peptide.
It has plenty of charged tryptophan tRNA and throws them in the leader peptide
and it slides on to 1 & 2 and 2 cannot form with 3 to form an antiterminator and
now 3 forms with 4 and you get the 3,4 termination stem loop. This is one of
those intrinsic terminators: high GC stem loop. Transcription is terminated early
before the 5 structural genes.
 When tryptophan levels are high you want to shut off the operon and then you
form the terminator stem loop intrinsic terminator.
 Summary: trp operon is regulated by 2 mechanisms, it has a corepressor, so when
there is plenty of tryptophan, tryptophan binds the trp repressor and then it can
bind the operator, blocking transcription; but if the transcript has already started
then it has a second mechanism of transcription attenuation where it can sense the
level of tryptophan and either terminate early or allow the transcription of the
entire operon.
Slide 39 - Control of Gene Expression
 Summary of prokaryotic transcription regulation
 We showed an example of the negative control of the lactose operon via a
repressor that binds the operator but can be induce by -galactosidase that can
bind to the repressor and inactivate it.
 In the tryptophan operon, we showed repression that needs a corepressor. In this
case, tryptophan will bind an inactive repressor making an active one that can
bind to the operator, thereby shutting down the operon.
 We also showed the positive control of the lac operon controlled by catabolite
repression where you have the CRP, which can bind the coinducer (in this case –
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cyclic AMP), so in glucose starvation, cyclic AMP levels rise and it activates this
inducer by binding to an upstream DNA binding sequence upstream of the
promoter thereby activating the loading of polymerase and transcription of the lac
operon.
This concludes the past 1.5 hours (first hour and half of second hour lecture) of
prokaryotic gene transcription.
Eukaryotic Gene Transcription
Slide 1 – Gene Expression Part 3
Slide 2 - Eukaryotic Vs. Prokaryotic Transcription
 Presence of a nucleus in eukaryotes
o means transcription & translation occur in separate compartments of cell
o all the translation occur outside in the cytoplasm
o in prokaryotes it could happen at the same time, as soon as you transcribe
a message ribosomes can get on there and start translating, and you can
have regulation like transcription attenuation with ribosomes sensing
tryptophan levels…not the same in eukaryotes
o in eukaryotes transcriptional machineries are in the nucleus and
translational machineries are in the cytoplasm
 Larger genomes (1000X between humans and E. coli)
o If you are 1000X larger you have 1000X more problems of locating genes
and determining where promoters are and 1000X larger problem of
regulation, how do you know when to turn certain genes on?
 Also have a problem of packing all of the DNA into the nucleus
o Chromatin structure in eukaryotes limits accessibility because it is so
tightly packed. How can you turn a gene on if it is so tightly packed?
 Three RNA polymerases (instead of just the single one in prokaryotes)
 Eukaryote pre-mRNAs are subject to extensive post-transcriptional modification
o There is a lot of processing. We have many introns in between exons in
eukaryotic genes.
Slide 3 - Genome Sizes of Various Organisms
 Note E. coli graph, that has a larger genome amongst bacteria
 Note humans/mammals
o The haploid genome has about 1000X more DNA than the E. coli genome
 However, humans have only 20 times as many genes as E. coli.
o 25,000 – 30,000 genes for eukaryotes
o E. coli has about 1000 genes
o 98.5% of the human genome is noncoding compare to only 11% of the E.
coli genome
 A lot of the E. coli genome coded for proteins
 In eukaryotes, only about 1.5% codes for proteins
Slide 4 - Eukaryotic Chromatin Structure
 The human genome contains 3 x 109 bp per haploid genome.
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How many base pairs in a diploid genome?
If the DNA of all 46 chromosomes from one cell were linked together, it would
measure one meter in length.
If the DNA in all cells of a single human was linked end to end, it would stretch
to the sun and back. A lot of DNA packed into a relatively small space.
How is the DNA packaged into the nucleus such that genes are still accessible to
the transcriptional regulatory proteins that control their expression?
Slide 5 - Characterization of Human Genomic DNA
 About 53% of the human genome is repetitive sequence
 A mixture of long interspersed nuclear elements, short interspersed nuclear
elements, retroviral-like elements, DNA transposon, duplications and single
sequence repeats
 The unique sequences represent most of the remainder of the genome
 In red here is shown actual protein coding sequences (1.5%)
 Introns, which are transcribed but don’t actually code for protein, are about 20%
of the genome
 There are unique sequences which aren’t transcribed in introns or exons but can
have regulatory sequences that control the expression of the genes.
Slide 6 - Eukaryotic Chromatin Structure
 Each eukaryotic chromosome contains a single linear (E. coli was circular)
supercoiled DNA molecule
o Not just naked DNA, but coded in nucleoprotein material
 Nucleoprotein material of the eukaryotic chromosome is called chromatin
(complex of DNA, protein and RNA)
Slide 7 – EM of a liver cell nucleus
 Note the cytoplasm, nucleus & nuclear membrane
 Two types of chromatin:
 Euchromatin – more accessible
 Heterochromatin – inaccessible, highly condensed DNA
 If you have a gene being expressed in this liver cell it would be in the
euchromatin region of the nucleus
Slide 8 - Eukaryotic Chromosome Structure
 Euchromatin - comprises most of the genome, transcriptionally active, susceptible
to DNaseI digestion (an enzyme that can cleave DNA)
 Heterochromatin - highly condensed inactive chromatin located at centromeres
and telomeres (less susceptible to DNase digestion)
 Centromere - attachment point for sister chromatids and spindle fibers
 Telomere - ends of chromosome
 These generalities. You can see exceptions.
Slide 9 - Telomeres and Telomerase
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What do you do on the ends of your chromosomes?
We have telomeres. There is a special enzyme called telomerase that is required to
maintain the end of our chromosomes.
Lagging strand DNA replication requires a primer and then polymerase uses that
primer to replicate the DNA.
What happens to the RNA primer at the end of the chromosomes? Once it gets
removed there is no DNA there. You are left with a shortened DNA on the end of
your chromosome. Every time the cell divides it gets shorter and shorter. In cells
such as stem cells, there this enzyme telomerase which is an RNA-dependent
DNA polymerase that adds this sequence of bases TTAGGG many times to the
end of the chromosome. In humans, it is about 1,000-1,700 of these sequences
The template for this is from an RNA molecule in the telomerase.
o The telomerase is a ribonucleo protein composed of two components:
 RNA component – the telomerase RNA has the template for the
repeating TTAGGG.
 Polymerase component
Now lagging strand synthesis it doesn’t matter if you have completely replicated
all of the DNA on the end because it is telomeric DNA and it can be shortened
without much consequence.
Know your telomerase!
What kind of cells express telomerase? (cancer cells are an example)
Slide 10 - Eukaryotic Chromatin Structure
 How do you pack all of the DNA into the nucleus?
 DNA associates with histones proteins to produce nucleosomes
 Nucleosome - the fundamental unit of organization of the chromatin fiber
 Each nucleosome contains a core particle of basic proteins – histones - which are
surrounded by DNA
 Electron microscopy shows fiber structure of chromosomes as “beads on a string”
o String = DNA; Beads = Histone
Slide 11 - Histones
 Small basic proteins, rich in lysine and arginine
o Basic because DNA is acidic
 Interact with DNA through electrostatic interactions
 Five major types of histones:
 H1, H2A, H2B, H3 and H4
 H2A, H2B, H3 and H4 form a complex of 8 proteins
 DNA is supercoiled around histone octets forming nucleosomes
 H1 associates with neighboring nucleosomes to form a more closely packed
structure 30nm solenoid
Slide 12 - Histone Structure
 Alpha-helical proteins
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Note H2A, H2B, H3, H4
3 alpha-helical domains called the histone fold
H2A and H2B can come together to form a dimer called a histone-handshake
(Figure C).
Have N-terminal tail where regulation can occur
Slide 13 - Histone Structure
 You can have two of these dimers that come together. Here you have H3
dimerizing with H4 to form the H3-H4 dimer; H2A and H2B come together to
form the H2A-H2B dimer.
Slide 14 - Histone Structure
 Two H3-H4s come together to form H3-H4 tetramer
 Same thing with H2A and H2B
 These two tetramers come together to form the histone octamer
 You can see the amino terminal tails look like they are flopping out in the breeze,
but there is a lot of regulation that can occur there
Slide 15 - DNA Wraps Around Histone Octomers
 The DNA helix makes 1.65 turns around the histone octamer.
 146 base pairs of DNA wraps around each histone octet.
 Histones are among the most highly conserved eukaryotic proteins.
o For example, the amino acid sequence of histone H4 from a pea and a cow
differ at only 2 of the 102 amino acids.
 Shows DNA double helix wrapping around the histone octet here shown in yellow
Slide 16 - Solenoid Formation: Role of H1 Histone
 Histone H1 is a globular histone that is highly conserved
 Very positively charged which helps compact the negatively charged DNA more
tightly around each histone
Slide 17 – Histone Tails
 Again, this is 1.65 turns. Histone H1 can come in and wrap it a little tighter and
can also bring neighboring histones in together to form more closed, compact
structures.
Slide 18 – Histone Tails
 Shows histone octet.
 You can see that even the amino terminal tails have structure to them, they are
associated with the DNA that is wrapped around these histones. They are not just
flopping around in the breeze.
 These histone tails can also interact with neighboring histones in DNA
 When these tails are acetylated, the interaction between nucleosomes are
diminished and the chromatin becomes more extended/open
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The lysines can be acetylated in these histone tails causing the histones to come
apart a little bit. When they are deacetylated, the neighboring histones can get
more closely packed.
Slide 19 - Solenoid
 The 30nm solenoid is pictured with a more compacted structure
 This picture shows the beads on a string model
 It gets further condensed in the presence of the H1 histone
 You can get it folded up into the solenoidal structure
Slide 20 – Eukaryotic Chromatin Structure
 Micrograph showing the solenoidal structure and the 10 nm DNA histones beads
on a string structure
Slide 21 – Eukaryotic Chromatin Structure – Looping and Miniband
 The solenoidal structure can be further condensed by looping and forming this
miniband structure.
 The 30 nm solenoid structure gets looped and attached to a protonation matrix and
these loops can be further condensed by wrapping them and ultimately you have a
fully condensed chromosome.
Slide 22 – Eukaryotic Chromatin Structure
 skipped?
Slide 23 – Eukaryotic Chromatin Structure
 Going from the 2 nm duplex DNA  10 – 11 nm beads on a string DNA wrapped
around histone  30 nm solenoid  looping structure  looping and folding
structure
 You can condense that meter of DNA into a very small space
 Once you are condensed like this in the metaphase chromosome, how do you turn
genes back on? That is what we will talk about tomorrow.
 Conclusion: we talked about prokaryotic transcription (which is simple to the
eukaryotic case) where you had a single polymerase with a sigma-factor
recognized promoter and you can have activators like CAP vs. tomorrow when
we talk about eukaryotic transcription it is much more complicated to identify
genes in all the DNA
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