Transcriptional Regulation in Prokaryotes

Simple terms: what is the difference between constitutive and inducible/repressible gene expression?

Constitutive is when genes are expressed in all conditions and inducible/repressible expression is expressed in SOME conditions (not all)

What kind of genes are expressed in constitutive expression?

Genes required for basic cellular functions (otherwise known as “housekeeping” genes)

ex: genes encoding RNA polymerase, ribosomal RNAs, ribosomal proteins, cytoskeletal components, and some metabolic enzymes

Where is inducible/repressible expression seen in single-cell organisms (prokaryotes, yeast)? What about for multicellular organisms (plants, animals)

Single-cell: Commonly in response to environmental and nutritional signals (certain genes expressed at certain times/conditions) (resource partitioning)

Multicellular: cell-cell communication during development, tissue-specific expression, or response to environment

What are two examples of levels of regulation of gene expression? Are they seen in both eukaryotes and prokaryotes?

Transcriptional (both) and posttranscriptional (mainly eukaryotes)

What does transcriptional regulation entail?

It can be seen in the initiation of transcription (chromatin structure and DNA methylation for eukaryotes + is very important in prokaryotes) and the shift from initiation to elongation

Why is transcriptional regulation so important for prokaryotes

Because it is their only way to regulate gene expression

Give examples of posttranscriptional regulation

Stability (half life) of mRNA, mRNA processing and transport to cytoplasm, efficiency of translation initiation, and stability +posttranslational modification of polypeptide

What are structural genes? what are they controlled by?

Genes that encode for proteins that function in metabolism. biosynthesis, or structural aspects of the cell

Controlled by regulatory genes

What are regulatory genes? (function + are they transcribed)

Genes that encode for regulatory RNAs or proteins (so they are transcribed) that control the expression of structural genes and/or their products (many have DNA-binding activity)

What are regulatory elements?

DNA sequences that are not transcribed but do assist with regulating other genes (commonly the site of binding of regulatory proteins)

Trans-acting vs cis-acting elements

Trans-acting elements are commonly regulatory proteins encoded by regulatory genes (ex: DNA binding proteins). A main difference versus cis-acting elements is that these elements can diffuse through the cytoplasm and act on target sites on ANY DNA molecule in the cell. Cis-acting elements, regulatory DNA elements/sequences that are the binding site for regulatory proteins, cannot move like trans-acting elements. These elements can only influence expression of adjacent genes on the SAME DNA molecules

What are the two general modes for transcriptional regulation? Compare and describe them

Positive and negative control

Positive control often entails a regulatory protein that is an ACTIVATOR that stimulates expression of target gene(s). Therefore, transcriptional activity is ON when positive regulatory protein is active, so these activator proteins will bind to target DNA sequences and activate transcription

Negative control involves a REPRESSOR that inhibits expression of target genes. If it is active, transcription is inhibited (if it is not active, transcription is occurring). There are multiple mechanisms involved in inhibition of transcription

Do positive and negative control occur in both eukaryotes and prokaryotes

Yes

What are effectors

Small molecules that bind to regulatory proteins and control their activity (not regulatory genes, proteins, or elements); they are responsive to the need for a specific metabolic pathway

What is inducible vs repressible expression?

Inducible expression: presence of effector enables transcription (transcription is often off until it is activated) (ex: lac operon)

Repressible expression: presence of effector inhibits transcription (transcription is on until repressed) (ex: trp operon)

What kind of enzymes does inducible expression control? What about for repressible expression?

Inducible is responsible for catabolic enzymes while repressible is responsible for anabolic enzymes

Explain how the lac operon is an example of inducible repression and how the trp operon is an example of repressible expression?

Lac operon: turned on when lactose is present

Trp operon: turned off when tryptophan is present

What goes on in:

1. negative inducible expression

2. negative repressible expression

3. Positive inducible expression

4. positive repressible expression

1. the repressor is inhibited by the effector so transcription is activated

2. the effector activates the repressor to allow for binding, inhibiting transcription

3. The effector activates the activator’s binding to regulatory sequence to allow transcription

4. The effector inhibits the activator from binding, so transcription is inhibited

How to determine whether something is positive/negative regulation? What about for repressible/inducible regulation?

Positive/negative: look at what regulatory protein is doing (seeing if it an activator=+ or repressor=-)

Repressible/inducible: look at whether transcription is activated (inducible) or inhibited (repressible)

How are bacterial genes organized?

Via operons

What are operons?

Set of functionally related structural genes transcribed from a SINGLE promoter

What are operators?

Cis-acting regulatory sequence where trans-acting regulatory proteins bind and affect transcription (this is where an effector binds)

It also coordinates the expression of multiple genes required for a particular pathway or function

In an operon regulated by a regulator protein, is the protein constitutively expressed on the operon?

no—it is expressed from a different gene (it is needed before the transcription of the operon)

Are regulatory genes expressed constitutively or repressible/inducible? Does it always bind the the operator

They are constitutively expressed, but whether or not it binds to the operator depends on the inducer or repressor molecules (effectors)

Why is lactose needed when glucose is the preferred energy source of E.coli and can be broken down via glycolysis? How is it used?

Lactose is an alternative carbon source to glucose (mainly used when we do not have glucose). In order to be used, the lactose must be actively transported into the cell and broken down into glucose and galactose

What are the three structural genes involved in the lac operon of E.coli? What are they responsible for?

LacY: permease

LacY: B galactosidase (B-gal)

lacA: encodes transacetylase (do not worry about this because the function is unknown)

What are the roles of permease and B-gal?

Permease transports lactose into the cell

B-gal breaks down lactose

What are the three aspects of the lac regulatory region?

lac promoter (lacP): binds RNA polymerase

lac operator (lacO): binds to the lac repressor protein

catabolite activator protein (CAP) binding site for CAP (CAP is trans acting why the binding site is cis acting)

Where does RNA polymerase bind in an operon? What about the regulatory protein?

RNA polymerase binds to the promoter

The regulatory protein binds to the operator

List all the genes present in lac operon

lac P: promoter

lacO: operator

lacY: permease

lacZ: B gal

What kind of regulation is the lac operon under?

It is inducible (activated when lactose is present) but it is under positive (activator) and negative (repressor)

Broadly, how is the lac operon negative and positive inducible expression

Negative: there is a lac repressor that inhibits lac transcription by binding to the lac operator

Postive: cAMP-CAP because if glucose is not present, it binds to the CAP binding site to induce transcription

In detail, how is the lac operson an example of negative pressible relgation? (Under what conditions)

In the ABSECNE of lactose, the repressor will bind to the lacO and bind the promoter

in the PRESCENCE of lactose, allolcactose (effector molecular + product to lactose being broken down) binds to the repressor and makes it unable to bind to the lacO, allow for transcription

Is there still B-gal and permease even without lactose? What about allolactose?

B-gal and permease can still be present from last time. When lactose initially enters via permease, B-gal can break it down to yield allolactose, resulting in induced negative expression

allolactose depends on lactose presence

What is an allosteric protein? How is the lac repressor an allosteric protein?

An allosteric protein is a protein whose shape is CHANGED when it binds to a particular protein. The lac repressor is an example because the binding of the inducer via binding site (allolactose or IPTG) results in a conformational (shape) change to the lac repressor

What is IPTG

Like lactose

What are the inducers of the lac operon?

Allolactose and IPTG because they result in the transcription (inducing of transcription) of the lac operon when binded to the repressor

What does it mean to partial diploid?

The organism is haploid for the majority of the genes but is diploid for some due to a plasmid. For example, E. coli is partially diploid because it has two copies of lac sequences: one from the plasmid and the other from the circular chromosome of E. coli

What can we investigate with partial diploids?

We can see how mutations affect the organism and the expresison of genes in prokaryotes

Who discovered/used partial diploids?

Jacob and Monod

lac- mutants cannot grow in what conditions?

When lactose is the only carbon source

Effect of mutation on function/cis or trans or NA/effect on lac mRNA expression: lacZ-

1. Nonfunctional b-galactosidase

2. NA

3. None (less of the enzyme to break lactose down)

Effect of mutation on function/cis or trans or NA/effect on lac mRNA expression: lacY-

1. Nonfunctional permease

2. NA

3. None (does not influence expression but there is less of the enzyme needed to transport lactose)

Effect of mutation on function/cis or trans or NA/effect on lac mRNA expression lacl-

1. defective repressor that cannot bind to lacO

2. trans (still produced but cannot bind)

3. Constitutive expression (no repressor present=transcription is not repressed)

Effect of mutation on function/cis or trans or NA/effect on lac mRNA expression: lacI^s

1. superrepressor (lacks binding site for inducer)

2. Trans

3. Non-inducible (will always repress transcription)

What does a superrepressor lack?

The binding site for the inducer

Effect of mutation on function/cis or trans or NA/effect on lac mRNA expression: lacO^c

1. fails to bind repressor protein/mutation with the operator

2. cis-acting (DNA sequence that affects adjacent genes)

3. Constitutive (because repressor cannot bind and repress transcription)

Effect of mutation on function/cis or trans or NA/effect on lac mRNA expression: lacP-

1. Defective prooter

2. cis acting

3. no expression/no lac structural genes can be expressed because RNA polymerase cannot even bind

what is the function of cAMP-CAP

cAMP-CAP binds to DNA near lacP at the CAP site to enhance the binding of RNA polymerase to lacP. There is a 50 fold increase in transcription as CAP activates expression of ~100 genes

What is the DNA binding domain in positive regulation of lac operon?

Helix turn helix

Describe positive regulation in detail with the lac operon (including high/low glucose environments)

High glucose: cAMP levels are low because adenylate cyclase is low. As a result, there is minimal transcription because of low cAMP-CAP

Low glucose: There is high adenylate cyclase, so there will be a high amount of cAMP. cAMP will then bind to CAP to form the cAMP-CAP complex. This will then bind to the CAP site to increase/activate transcription of lac operon

Is lactose needed when glucose levels are high? (explain how it relates to cAMP)

Not really since glucose is the preferred energy source (hence why cAMP levels are low when glucose is high)

Describe whether the operon is off or on in

1. High glucose and lactose

2. High glucose and low lactose

3. Low glucose and low lactose

4. Low glucose and high lactose

1. no cAMP-CAP binding; even though repressor is not bound, there is minimal transcription without cAMP-CAP binding

2. no cCAMP-CAP is produced but the repressor is also on=closing

3. high cAMP-CAP is present on CAP site but repressor is bound=operon is off

4. high cAMP-CAP on CAP site to enhance transcription + no repressor is bound to activator=operon is on

What are the five structural genes required to synthesize tryptophan?

trpE, trpD, trpC, trpB, and trpA

What kind of expression is the trp operon?

It is negative repressible, so trp structural genes are only expressed in the absence of tryptophan/when tryptophan levels are low (repressor involved→ negative and transcription is inhibited →

Is the trp operon expressed when

1. tryptophan is present

2. tryptophan is not present

1. If tryptophan IS present, the genes are NOT expressed

2. If tryptophan is NOT present, the genes ARE expressed

What are the two levels of control of trp gene expression?

1. trp repressor (trpR) will act on trp operator (trpO)

2. attenuation (do not need to know)

For trp, how fast is the response? and what does the response help with?

The response is extremely rapidly, and it is used in protein synthesis

Describe in detail how the trp operon works in low and high levels of tryptophan

Low: the trp repressor cannot bind to the trp operator, so the trp structural genes are transcribed—tryptophan is produeced (but it is needed since tryptophan is low)

High: the trp repressor binds with tryptophan to activate it, allowing it to bind to the trp operator. As a result, the expression of trp structural genes is repressed

What is the effector and co-repressor of the trp operon?

Tryptophan (it will work with the repressor to repress—hence why it is called a co-repressor)

How is the trp operon logical?

Because it only puts energy to produce tryptophan when it needs it; when tryptophan levels are high, no more tryptophan is made. when tryptophan levels are low, more tryptophan is made (activation of trp operon) plus there is not any tryptophan to act as a repressor