Transcriptional regulation - Prokaryotes (18)

Question 1

Why is transcription regulated? Why aren’t all the genes transcribed at all places at the same time?

Answer 1

All cells of biological systems have the same genome and basal transcription machinery. In principle all genes could be expressed at any time, but this is not the case.

Different kinds of cells exist to perform very different functions, and to perform these functions they need to express specific sets of genes. Two cells with identical genomes can transcribe different genes in order to adapt to their environment or achieve different functions.

Examples:

• E.coi will express different genes depending on carbon source - either glucose or lactose
• Regulation of transcription allows development and functions of different cell types (tissues) in metazoans , e.g. nerve cells, cardiac cells, muscle cells

Question 2

Housekeeping genes

Answer 2

Genes that are transcribed all the time because they are involved in maintaining basic cellular functions (translation, energy, etc.) to keep the cell alive.
These genes do not (or weakly) depend on regulators because the basal transcription machinery is sufficient.

Question 3

Regulated genes

Answer 3

Genes that are transcribed in response to specific intra- or extracellular signals like hormones, growth factors, UV, etc.
Transcribed in a given cellular type like neurons, muscle, nerves etc.
These genes highly depend on transcriptional regulators.
If you compare the same chromosome in different cell types, you will see different specific genes light up in each cell type.

Question 4

Regulation of transcription during development of metazoans

Answer 4

Both in prokaryotes and eukaryotes regulation of genes must occur at the right place at the right time (and also regulation from day to day). While transcriptional regulation in prokaryotes is relatively complex, it is highly complex in metazoans.
Cellular (tissue) identity is determined by the timely and localized transcription of specific genes during development. Transcriptional regulation defects lead to dramatic alterations of development, e.g. legs growing on Drosophila’s head.

Question 5

Basal (general) transcription machinery

Answer 5

Consists of RNA polymerases + initiation factors, and is required for the transcription of all genes (without polymerase, there is no transcription). RNAP binds to core promoter.
Prokaryotes: RNAP core + sigma-factor
Eukaryotes: RNAPs + general transcription factors (GTFs)

Question 6

Transcriptional regulators (as opposed to basal transcription machinery)

Answer 6

Usually don’t bind to the core promoter, but to regulatory sequences that can be located near the core promoter or very far away. Bind to specific genes at specific regulatory sequence.

These regulators integrate an intra- or extracellular signal (hormone, sugar, growth factor, UV, differentiation, cell cycle, starvation etc.) -> Regulators triggered by these signals bind to the DNA, and in turn regulate the basal transcription machinery.

Linear representation: Signal -> Regulator -> Basal machinery -> Regulation of transcription

Question 7

Regulatory proteins (which step do they affect + types)

Answer 7

Regulators mostly affect the initiation step, and therefore the number of RNA copies. Why? Regulating initiation is the most energetically efficient way, because it costs less to regulate one gene than thousands of already synthesized mRNAs or translation. Don’t have to cope with the mess you already made.

Transcriptions can be:

• ACTIVATORS that will stimulate transcription. A gene under the activity of an activator will give rise to a lot of corresponding RNA proteins.
• REPRESSORS that will prevent transcription. A gene under the activity of a repressor will barely be expressed.

Question 8

What are the three ways regulators can act on intiation of transcription?

Answer 8

1. Model the binding of the basal transcription machinery to the promoter
2. Affecting the promoter-bound RNAP by allostery
3. Regulators acting from a distance (DNA loops)

Question 9

How do regulators act on initiation of transcription? 1

Activators and repressors model the binding of the basal transcription machinery to the promoter

Answer 9

Regulators can regulate association of the basal machinery to the promoter.

For some housekeeping genes RNAP may bind some promoters spontaneously/weakly, featuring a basal level of transcription (if initiation factors find a promoter, they will probably start transcribing a little bit).

Other genes are controlled really tightly by repressors and activators. Repressors can bind to OPERATORS (overlapping with promoter) that prevent RNAP binding to promoters (compete for binding to promoter) - no transcription.
Activators can bind to their binding site (upstream) and recruit and stabilize the polymerase (recruits RNAP and GTFs and helps them initiation transcription) - cooperative binding, high levels of transcription.

Question 10

How do regulators act on initiation of transcription? 2

Regulators affecting the promoter-bound RNAP by allostery

Answer 10

RNAP can bind very efficiently to some promoters but remains inactive. The close complex is very stable, so there is no spontaneous isomerization -> No transcription. It needs an activator to trigger open complex formation.
These activators trigger conformational changes of RNAP by allosteric regulation -> Bind to one side and triggers conformational change on the other side -> Formation of open complex.
On the contrary, some repressors use allostery to inhibit formation of an open complex -> Binding of repressor alters the active site of the RNAP to be unable to bind to the substrate.

Question 11

How do regulators act on initiation of transcription? 3

Regulators acting from a distance (DNA loops)

Answer 11

Regulators and regulated proteins can bind far away from each other (tens of Kbs away) -> Triggers formation of a DNA loop. The activator bound to its binding site contacts the basal transcription machinery via the contact of a DNA/chromatin loop. Sometimes there are DNA-bending proteins that facilitate/stabilize the formation of DNA loops so that initiation occurs.
This feature doesn’t occur very often in prokaryotes (few and short DNA loops), but very often in eukaryotes (numerous and long DNA loops).

Question 12

Prokaryotes: What is a famous and well-studied example of regulation in prokaryotes?

Answer 12

The lac operon, discovered in the 1960s.

Equivalent to discovery of self-regulation of the genome (genes encoding regulators -> feedback loops).

Question 13

Prokaryotes: What is the purpose of the lac operon in E.coli? Background

Answer 13

The need to adapt to available carbon sources.

Background: Jacque Monod discovered that in a medium with both glucose and lactose, the bacteria showed that the bacteria had two phases of growth (diauxic growth). Hypothesis - first they metabolized glucose (easier), and when the glucose runs out -> Lag phase where they start expressing the genes involved in the metabolism of lactose -> Lactose can be used, the second phase of growth starts.

Question 14

Prokaryotes: Operon

Answer 14

One promoter controlling the expression of several genes (specific to bacteria). All the genes are transcribed into a single mRNA, called a polycistronic mRNA. The ribosome translates this mRNA strand into several proteins.
Example: The lac operon

Question 15

Prokaryotes: The structure of the lac operon

Answer 15

Composed of:

• The 3 lac genes (lacZ, lacY & lacA) are sequentially located next to each other forming the lac operon
• The lac promoter controls the transcripton of the 3 genes into a unique polycistronic mRNA

In addition to the binding site for the sigma factor, it contains a CAP site and an operator, which are binding sites for regulators.

Question 16

Prokaryotes: The lac genes (names + function)

Answer 16

The three lac genes are called lacZ, lacY and lacA and are sequentially located next to each other.

mRNA translated into 3 proteins:

• lacZ encodes the beta-galactosidase -> Cleaves lactose into glucose + galactose (easier to metabolize)
• lacY encodes the lactose permease -> Transports lactose into the cells (internalizes lactose from the medium)
• lacA encodes the thiogalactoside transeacetylase -> Rids toxic thiogalactosides that enter the cells with lactose

Question 17

Prokaryotes: Regulatory proteins involved in the lac operon

Answer 17

The lac promoter contains two regulatory sequences: The CAP site and the operator.

• The activator CAP (Catabolite Activator Protein), also known as CRP, binds to the CAP site
• The Lac repressor binds to the operator

Question 18

Prokaryotes: Regulation of lac genes by CAP and Lac repressor

Answer 18

Each regulator mediates a signal (the amount of glucose/lactose in the medium) that regulates transcription.

Lac repressor mediates the signal “absence of lactose”. No lactose present (but glucose present) -> No transcription of the lac genes.

CAP mediates the signal “absence of glucose”. No glucose present (but lactose present) -> Transcription of the lac genes.

When both glucose and lactose are present in the medium, no regulators are bound, and there is a basal level of transcription.

Question 19

Prokaryotes: How do CAP and Lac repressor control RNAP binding to promoter in opposite ways? How do they respectively recruit or prevent recruitment of RNA polymerase and the sigma factor to the promoter?

Answer 19

The Lac repressor and RNAP compete for the promoter. Operator overlaps with RNAP binding region, and bound lac repressor prevents RNAP from loading onto the promoter.

CAP and RNAP bind cooperatively. Normally, when there is no UP-element and weak -35 element at the lac promoter, there will be a weak RNAP binding. Once it is bound to the CAP site, the CAP activator will recruit the sigma factor and RNAP -> Promoted binding.

Question 20

Prokaryotes: How is RNAP recruited by CAP?

Answer 20

CAP consists of a DNA binding domain (DBD) and an activation domain (AD). CAP serves as an UP element:
CAP binds to the CAP site as a homodiner via the DBD (recognizes site) -> Interacts directly with alpha-CTD of RNAP (aka. CAP serves the function of an UP element, which is lacking from the lac operon) -> Stabilization of RNAP on promoter -> Transcription

Question 21

Prokaryotes: How do the CAP and lac repressor bind the DNA in a similar manner, despite having opposite functions?

Answer 21

Regulators usually bind to the DNA as a homodimer at inverted repeat sequences. Each repeat or half-site is a binding site for one monomer that contains a helix-turn-helix motif.

• 1 alpha-helix binds the DNA major groove in a sequence-specific manner (recognition helix)
• 1 alpha-helix interacts with the DNA backbone

Question 22

Prokaryotes: Leaky transcription (definition + purpose)

Answer 22

Leaky transcription of the lac operon = low expression of lactose metabolism enzymes. Basal levels of transcription - when RNAP is around, it can weakly bind to promoters and trigger transcription. Lactose pearmease allows entry of lactose when present (internalizing lactose from the medium), and beta-galactosidase converts lactose into allolactose (functions as allosteric effector) via transglycolation.

Purpose? If lactose is suddenly in the medium, the bacteria want to be able to metabolize it before it can activate the operon. Leaky transcription makes a small amount of the different enzymes that can prime the process.

Question 23

Prokaryotes: How do glucose and lactose signal to the regulators? 1
Allosteric regulation of Lac repressor by lactose signaling

Answer 23

In the absence of allolactose, the lac repressor is bound to the operator -> No transcription of the lac operon.

Lactose in the medium -> Thanks to leaky transcription (of lactose permease and beta-galactosidase), there will be a few molecules of lactose converted into allolactose (functions as an allosteric effector) present within the cell.

Allolactose binds to Lac repressor -> Allosteric effect -> Change conformation of DBD (DNA-binding domain) -> Repressor no longer able to bind to the operator on the DNA -> Full transcription of the lac operon.

Question 24

Prokaryotes: How do glucose and lactose signal to the regulators? 2
Allosteric regulation of CAP repressor by glucose signaling

Answer 24

Absence of glucose activates CAP. Glucose inhibits the enzyme adenylate cyclase that converts ATP into cAMP (cyclic AMP)

• High glucose levels = low cAMP levels
• Low glucose levels = high cAMP levels

cAMP is an allosteric effector of the CAP activator. When cAMP binds to the CAP activator, it will change the conformation of the DBD (same mechanism as for the repressor). Allosteric effect -> Change of conformation of DBD -> Binding to CAP site -> Full transcription of lac operon.

Before binding of cAMP, there are two helices in CAP which are unable to bind to the DNA -> Upon cAMP binding, their orientation changes so that they’re able to align with the DNA major groove at the CAP site.

Question 25

Prokaryotes: How has the lac operon been used as a tool by molecular biologists? (lab)

Answer 25

You can engineer the lac operator to mass produce recombinant proteins.

1st phase: We have E.coli genome in which the gene encoding for a polymerase (T7, coming from bacteriophage) has been inserted. These genes can be transcribed into mRNA coding for the protein T7, but they are under control of the lac repressor. We give the cells a molecule that mimicks lactose -> IPTG -> Works like allolactose, prevents repressor to bind to DNA -> Enable transcription of the T7 protein (not the lac operon). In other words, the genome is engineered so that IPTG induces ecpression of T7 polymerase.

2nd phase: (Parallelly) Introduction of a plasmid in which there are two important things: The promoter to which the T7 polymerase can bind to, and a gene of interest (under control of the promoter), allowing you to encode the recombinant protein you want to mass produce.

IPTG added to the cells -> Trigger expression of T7 polymerase -> Goes to the T7 promoter in the plasmid -> Triggers transcription of inserted gene -> Translation -> Gives rise to massive amounts of protein. Overexpressed protein band popping up on SDS page in presence of IPTG.

Question 26

Prokaryotes: Mention 4 other regulatory mechanisms

Answer 26

• Cascade of alternative sigma-factors in response to heat shock
• NtrC’s activation by allostery
• MerR’s activation by allostery
• AraC regulating araBAD operon

Question 27

Prokaryotes: Alternative sigma-factors direct recruitment of RNAP to alternative promoters

Answer 27

While o^70 (which binds to -10 and -35) is the main sigma-factor in bacteria, it is mostly involved in transcription of housekeeping genes.

Other sigma-factors are mobilized in response to signals to direct RNAP to genes regulated by these signals.

• o^32 is expressed during heat shock and directs RNAP to genes responding to heat shoch that have specific promoter sequence.
• o^70, o^28 and o^34 are involved in early, middle and late genes expression during infection by bacteriophage SPO1, respectively
• o^54 directs NAP to genes involved in nitrogen metabolism

Cascade of gene expression (regulation through usage of alternative sigma-factors)
Infection by bacteriophage SPO1 triggers 3 waves of gene expression: Early genes, middle genes and late genes (relative to time of infection). o^70 triggers transcription of early genes, among early genes there’s one encoding o^28 which triggers the transcription of middle genes, among middle genes there will be one encoding o^34 which trigger transcription of late genes.

Question 28

Prokaryotes: NtrC (and glnA)

Answer 28

NtrC are activators which regulate the basal transcription machinery via allostery. Specifically activate genes involved in nitrogen metabolism, especially glnA.

When the levels of nitrogen gets low -> Cells need to keep the expression of glnA up -> NtrC is triggered and bind to a segulatory sequence (150 bp upstream of promoter) -> Interacts with o^54 -> Creates a DNA loop.
ATPase activity of NtrC -> Binding of NtrC creates an allosteric change in the structure of the complex, which triggers the conformation of the open complex -> Transcription of glnA.

When o^54 is bound to the glnA promoter, it cannot trigger formation of the open complex spontaneously by isomerization, but needs the allosteric change triggered by the activator.

Question 29

Prokaryotes: MerR (and merT)

Answer 29

merT is a gene involved in response to mercury (Hg^2+) - mercury in the medium, the cell needs merT to cope with this.
In absence of mercury the activator MerR bound to the merT promoter.

• No Hg^2+ -> MerR remains -> -10 element not aligned properly with -35 element -> o^70 can bind but can’t activate RNAP
• Hg^2+ > MerR conformational changes -> DNA twisting -> Correct alignment of -10 with -35 element -> o^70 can recruit RNAP -> Transcription of merT

Activator already bound, presence of signal triggers allosteric change which leads to recruitment of the basal transcription machinery.

Question 30

Prokaryotes: AraC regulating araBAD operon

Answer 30

This operon responds to the levels or arabinose in the medium. araBAD is under control of the activator AraC.

• No arabinose -> AraC binds as a homodimer to the sequence called I1 and araO2 (forms a DNA loop) -> No RNAP recruitment
• Arabinose present -> Allsoteric change of AraC -> Dimer now binds exclusively to binding sites I1 and I2 -> Recruitment of RNAP -> Transcription of araBAD genes.

Activator already bound, presence of signal triggers allosteric change which leads to recruitment of the basal transcription machinery.