L9-Regulation and signaling

What are the two very general modes of protein regulation in E. coli?

protein activity and protein amount

Describe protein activity

activate or inactivate a protein that’s already made 

Describe protein amount

control the synthesis or degradation of a protein

Protein activity regulation can be categorized as ___.

post-translational regulation

What are examples of protein regulation that fall under the category of protein activity regulation?

Covalent modification, Allosteric Regulation & Feedback Inhibition, Proteolytic Cleavage, Allostery by regulatory sRNAs

What are examples of covalent modifications?

Adenylation, Phosphorylation, Methylation, Uridylation, Acetylation

How does the covalent modification, Phosphorylation/Dephosphorylation, work?

Enzymes called kinases add a phosphate group (typically from ATP) to specific amino acid residues (serine, threonine, or tyrosine), while phosphatases remove it. The addition of a bulky, negatively charged phosphate group alters the protein's tertiary structure, which can either activate or deactivate an enzyme, or create a binding site for protein-protein interactions.

How does Allosteric Regulation & Feedback Inhibition work?

Small regulatory molecules (effectors) bind non-covalently to an allosteric site—a region distinct from the protein's active site. This binding induces a conformational change that either increases affinity for a substrate (positive allostery/activation) or decreases it (negative allostery/inhibition). In metabolic pathways, this often manifests as feedback inhibition, where the end-product of a pathway inhibits the first committed enzyme to prevent overproduction.

How does Proteolytic Cleavage work?

Some proteins are synthesized as inactive precursors called zymogens (or proenzymes). They are activated through the irreversible, targeted cleavage of one or more peptide bonds by another enzyme, causing the protein to fold into its active conformation.

Any step in either transcription or translation can be regulated to alter ___.

protein amount

Protein amount regulation can be categorized as __.

transcriptional and posttranscriptional regulation

What are examples of protein regulations that falls under the category of protein amount regulation?

transcriptional control, translational control, proteolysis, and inhibition of translation by regulatory sRNAs

What are some examples of transcriptional control?

DNA Topology, Promoter recognition, Transcriptional repression, transcriptional activation, transcriptional enhancement, transcription termination, regulatory RNAs (sRNAs) → transcription initiation/termination.

How does DNA topology relate to transcriptional control?

An effective way to control the initiation of transcription is by changing the DNA topology to either ­ prevent or promote σ -­ R NAP binding to the promoter. Most promoters are, for example, sensitive to the degree to which the DNA is supercoiled or packed. In fact, cells devote many proteins to the control of the topology of their DNA.

What are some examples of posttranscriptional control?

Translational control (via translational repression), inhibition of translation by bacterial small RNA (sRNA), mRNA stability, proteolysis.

How does mRNA stability relate to posttranscriptional control (protein amount)?

By altering the stability of mRNA, a cell can regulate the amount of its protein products. For example, upon binding, some sRNAs trigger the degradation of their target mRNA molecules .

What are two examples of transcriptional repression (a type of protein amount regulation)?

The Lac Operon Repression and the Trp Operon repression.

Which type of regulation is most common in bacteria?

Transcriptional Control is the most common type of regulation in bacteria. By preventing the synthesis of the mRNA transcript, neither the encoding RNA nor the protein is made; thus resources are not wasted.

Which type of regulation could occur only in prokaryotes?

Operon-based regulation and attenuation

Which type of regulation would involve a sigma factor?

transcriptional regulation (specifically, transcription initiation) of bacterial genes. This is a type of protein amount regulation.

Which type of regulation would involve a DNA bending protein?

DNA bending protein like topoisomerases are involved in modulating supercoiling and DNA topology. DNA topology is a transcriptional control and is a type of protein amount regulation.

Which type of regulation would involve a small antisense RNA?

Antisense mechanism of translational control by sRNAs. This is a protein amount regulation

Which type of regulation would involve an RNase?

RNase is used in RNA degradation where RNA molecules like mRNA are broken down, which is part of post-transcriptional control (mRNA stability). This is a regulation of protein amount.

Which type of regulation would involve a protease?

Protease is involved in cleavage and proteolysis, which are post-translational controls. This primarily is a regulation of protein amount, but can also be a regulation of protein activity because it can remove a protein that regulates other proteins.

Which type of regulation would involve a transcriptional repressor?

A transcriptional repressor is involved in transcriptional repression control, which is a type of protein amount regulation.

Which type of regulation would involve a kinase?

A kinase is involved in covalent modification. It transfers phosphate to an acceptor molecule. The opposite enzyme of a kinase is phosphatase. Covalent modification is a type of post-translational control and a protein activity regulation. 

Describe generally what the mechanism of attenuation is

During attenuation the majority of transcripts are prematurely terminated shortly after ­ transcription initiation. This pausing during transcription is typically prompted by formation of a hairpin/stem loop in the nascent RNA chain.

What is the purpose of the genes in the trp operon?

The trp operon has the trpE-A genes encoding enzymes for tryptophan synthesis

What does the trpL gene encode for?

it encodes the trp Leader peptide (trp mRNA), which is 14 amino acids long and located upstream of trpE-A in genome

What are the two important properties of the transcript (mRNA) of trpL?

1. It can form 2 different hairpin/stem-loop structures

• Terminator (short): causes premature transcription termination 

• Antiterminator (long): does not cause premature transcription termination

2. Has 2 adjacent tryptophan codons (UGG UGG). These 2 adjacent tryptophan codons serve as “sensing codons” of tryptophan availability. 

Are the trpE-A genes of the trp operon expressed during conditions of trp scarcity or abundance? Why?

The trpE-A genes are only expressed during conditions of trp scarcity, because tryptophan is an energetically expensive amino acid to produce, requiring significant ATP and reducing equivalents. When tryptophan is already abundant in the environment, synthesizing it would waste valuable cellular resources. Conversely, when environmental supplies drop, the cell must activate these genes to survive and continue protein synthesis.

How do the mRNA, leader peptide, RNA polymerase, and ribosomes orchestrate attenuation when it is needed?

When tryptophan is abundant, tRNAs charged with trp are abundant so the ribosome doesn’t stall at the trpL’s trp codons on the mRNA. This prevents the anti-termination configuration from forming on the mRNA. The mRNA adopts instead a terminator conformation that pauses the RNA polymerase (RNAP) and then prematurely terminates transcription of the operon.

What do mRNA, leader peptide, RNA polymerase, and ribosomes when attenuation is not needed?

When tryptophan is scarce, the ribosomes stalls at­ these tryptophan codons of the trpL mRNA, waiting for the scarce tryptophanyl-­ tRNAs. The mRNA is then elongated and acquires the anti-termination configuration so RNA polymerase (RNAP) will continue transcribing the enzymes for making tryptophan.

How is an operon different from a regulon?

A regulon is a set of operons controlled by a common regulator.

An operon is a contiguous group of related protein-­coding genes that are transcribed together under­ the control of a single regulatory site (promoter).

What is a modulon?

A modulon is a set of regulons controlled by a common regulator (such as CAP-cAMP for glucose-mediated catabolite repression)

Describe catabolite repression

a common regulator (CAP-cAMP) controls transcription of many different catabolic enzymes, so bacteria selectively use their preferred energy source first.

What is the relationship between glucose levels and cAMP levels?

They are inversely related to each other.

Describe how the lac operon is regulated in conditions with and without lactose, and with and without glucose.

When lactose is present, the inducer will remove the LacI repressor, however, RNAP will not be activated and begin transcription of the lac operon, because it has not been stimulated by CAP-cAMP to help it clear the promoter. CAP-cAMP will not be available until all the glucose is exhausted from the medium because the protein system that transports glucose (Pts) inhibits the enzyme (adenylate cyclase) that makes cAMP. As long as glucose is pres­ent, the adenylate cyclase is inactivated and the levels of cAMP decreased. But once glucose was used up, cAMP was made, and CAP-cAMP became available, the RNAP can be stimulated by it and the lac operon can be transcribed to use the available lactose.

What is the purpose of the lacZ (Beta-galactosidase) in the lac operon?

Cleaves the disaccharide lactose into its constituent monosaccharides, glucose and galactose. It also plays a critical secondary role by isomerizing a small fraction of lactose into allolactose, the true intracellular inducer of the operon.

What is the purpose of the lacY (Lactose permease) gene in the lac operon?

A transmembrane transport protein embedded in the inner cell membrane that actively pumps lactose from the extracellular environment into the cytoplasm.

What is the purpose of the lacA (Galactoside transacetylase) gene in the lac operon?

An enzyme that transfers an acetyl group from acetyl-CoA to beta-galactosides.

How is the the Lac Repressor regulated?

Regulation: Encoded by the regulatory gene lacI (which sits nearby and is expressed constitutively at low levels).

Mechanism: In the absence of lactose, the repressor binds tightly to the operator, blocking transcription. When lactose enters the cell, it is converted into allolactose, which binds allosterically to the repressor. This changes the repressor's shape, causing it to fall off the operator DNA.

How is Catabolite Activator Protein - CAP regulated?

Regulation: Regulated indirectly by glucose levels via intracellular cyclic AMP (cAMP). When glucose is abundant, intracellular cAMP levels drop. When glucose is scarce, adenylyl cyclase synthesizes high amounts of cAMP.

Mechanism: cAMP binds to CAP, forming an active CAP-cAMP complex. This complex binds to the CAP-binding site on the DNA, bending it to aggressively recruit RNA polymerase and stimulate transcription.

What are the relevant control regions in the DNA of the lac operon?

The CAP-Binding Site: A DNA sequence located just upstream of the promoter where the catabolite activator protein binds to enhance RNA polymerase recruitment.

The Promoter (P): The specific nucleotide sequence where RNA polymerase binds to initiate transcription of the structural genes.

The Operator (O): A DNA sequence overlapping the promoter where the lac repressor protein binds. When the repressor is parked here, it physically blocks RNA polymerase from moving forward, halting transcription.

Under which conditions would lac repressor and CAP-cAMP bin to DNA and lac operon genes be expressed?

Lactose	Glucose	Repressor	CAP–cAMP activation	lac operon expression
Absent (-)	Present (+)	Bound to operator	Absent	Essentially off
Absent (-)	Absent (-)	Bound to operator	Present	Essentially off—the repressor still blocks transcription
Present (+)	Present (+)	Released from operator	Absent	Low (off)
Present (+)	Absent (-)	Released from operator	Present	High

Why is there a lag phase for e.coli to switch from using glucose to lactose?

It takes time for adenylate cyclase to be activated, for cAMP to be made and bind CAP, and for the cAMP-­ CAP complex to bind and stimulate the RNAP at the lac operon.

What type of signaling mechanism do most bacteria use to sense and respond to external signals?

The primary signaling mechanism that bacteria use to sense and respond to environmental changes is the two-component regulatory system (TCS).

Describe how two-component regulatory system (TCS) works?

Sensor Histidine Kinase (HK): Typically an integral membrane protein (homodimer) with an extracellular sensing domain and an intracellular kinase domain. When a specific environmental stimulus (such as a change in oxygen, nutrients, pH, or osmolarity) is perceived, the kinase undergoes a conformational change and autophosphorylates by transferring a phosphate group from ATP onto a conserved histidine residue within its own structure.

Response Regulator (RR): A cytoplasmic protein containing a receiver domain and an effector (usually DNA-binding) domain. The sensor kinase then transfers this phosphate group from its histidine residue onto a conserved aspartate residue on the response regulator's receiver domain. By binding DNA to turn genes on or off.

What are overview steps of two-component regulatory system (TCS)?

1. Signal Detection: The extracellular or membrane-spanning domain of the Sensor Histidine Kinase binds to a specific environmental trigger or stressor

2. Auto-Phosphorylation: Binding causes a conformational change that activates the kinase domain. The HK uses ATP to transfer a phosphate group onto a conserved histidine residue within itself

3. Phosphotransferase (Signal Relay): The HK transfers the high-energy phosphate group from its histidine residue to a conserved aspartate residue located on the receiver domain of the cognate Response Regulator

4. Cellular Response: Phosphorylation activates the output/effector domain of the Response Regulator (often a transcription factor). The active RR changes its affinity for target DNA sequences, upregulating or downregulating specific sets of genes (regulons) needed for the new condition 

5. Reset/Dephosphorylation: Once the environmental stimulus fades, the sensor kinase or associated phosphatase enzymes remove the phosphate group from the response regulator, returning the system to its baseline, inactive state.

What is the most common output of two-component signaling mechanisms in bacterial cells?

The most common output of a two-component signaling cascade is transcriptional regulation (altering gene expression). Once phosphorylated, the response regulator undergoes a structural shift that enhances its affinity for specific promoter regions on the bacterial chromosome. Depending on the system, this binding either activates or represses target genes, allowing the cell to rapidly synthesize proteins tailored to the new environment. (Note: Some systems can also directly influence cellular behavior, such as controlling flagellar motor rotation for chemotaxis).

Describe how FixL/J function in rhizobia’s two-component regulatory system.

FixL/J in Sinorhizobium meliloti is a two- component system that controls transcription in response to O2 concentration.

FixL/J activates transcription of the gene for nitrogenase, which catalyzes nitrogen fixation when [O2] is low.

Nitrogenase enzyme is inactive when [O2] is high, so it would be a waste to make it then.

Describe how ExoS/ChvI function in rhizobia’s two-component regulatory system.

ExoS/ChvI is a two- component system in Sinorhizobium meliloti that controls transcription in response to an unknown signal.

ExoS/ChvI activates transcription of genes for polysaccharide production.

The right amount of polysaccharide is needed to form the nitrogen-fixing symbiosis with plants.

What would be the effect of a mutation that inactivated a sensor-kinase? (for example, mutation of the histidine that is the site for phosphorylation in ExoS?)

No gene expression of polysaccharide production

What would be the effect of a mutation that made a response regulator be constitutively phosphorylated? (for example, mutation of the aspartate in ChvI so that ChvI appeared to be always phosphorylated?)

Too much gene expression of polysaccharide production