Slide 15 General Organization of Operons

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.
Picture here is in your book showing 3 structural genes
Has an upstream promoter labeled “P”
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
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
lacZ – codes for Beta-Galactosidase
lacY – codes for a membrane protein called Permease
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
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
DNA binding in the N-terminal domain
Inducer binding at the C-terminal domain

Slide 20 - lac OperonlacI 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
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

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.
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.
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
If you have glucose around, the cell is happy metabolizing glucose, it won’t turn on the lac operon
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?
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
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
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
No lactose will cause the lac repressor to bind to the operator and shut down the operon.
- Glucose, - Lactose
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
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.

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”
If you have plenty of tryptophan you don’t need to make these enzymes and you shut it down
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
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:
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.
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.
1,2; 3,4 or 2,3.
If the 2,3 is formed, it is the antiterminator and you will get expression of the downstream genes.
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 – 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
means transcription & translation occur in separate compartments of cell
all the translation occur outside in the cytoplasm
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
in eukaryotes transcriptional machineries are in the nucleus and translational machineries are in the cytoplasm
Larger genomes (1000X between humans and E. coli)
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
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
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
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.
25,000 – 30,000 genes for eukaryotes
E. coli has about 1000 genes
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