MIP 443 (Secretion Systems - Riboswitches)

Which secretion system is calcium dependent, and why does it require it?

T1SS; proteins are secreted unfolded, and folding is induced extracellularly by the binding of calcium

What are substrates of T1SS?

Repeats-in Toxins(hemolysins) that rupture host cell membranes; adenylate cyclases, lipases, proteases, etc.

Which secretion system uses a pseudopilus, and do the substrates need to be folded or unfolded before secretion?

T2SS; substrates need to be folded (folded in the periplasm if exported with Sec)

What are substrates of T2SS?

Proteins involved in adaptation, virulence, pathogenicity(virulent strains), environmental adaptation enzymes(non-virulent)

Which secretion system injects proteins directly into host cells to increase colonization and survival?

T3SS

Which secretion system requires ATP and PMF, and secretes substrates that lack a signal sequence?

T3SS

Functions/substrates of T4SS?

Conjugative transfer of DNA; DNA uptake/release; secretion of effector proteins or DNA/protein complexes into other cells

Which secretion system is found in both G+ and G- and loads unfolded substrates to secrete?

T4SS

List domains of T5SS and how it functions to transport substrates

Alpha-domain = passenger, beta-domain = transporter. First, the unfolded transporter is transported to periplasm by SecYEG; chaperones stabilize (but not fold); transporter domain is inserted into OM with help of BamA; transporter domain folds into a beta-barrel; unfolded alpha domain passes through the transporter, folds into a beta-helical structure

Which secretion system is energy independent?

T5SS

Which secretion system secretes toxins into other bacteria as a competition mechanism?

T6SS - contact dependent!

Three basic levels of regulation

1. Gene expression (transcription)

2. Protein production (translation)

3. Protein function (enzyme activity)

First step of transcription

RNA polymerase recognizes and binds the promoter

Where do activators and repressors bind, and how are they turned on/off?

They bind promoter regions of genes(transcription), and they can be turned on or off by binding small molecule metabolites or by post-translational mod.

Explain the lac operon (and what kind of gene expression it is)

Lac operon is inducible - gene expression is induced when glucose is absent and lactose is present. Transcriptional level, alters rate of enzyme synthesis

Explain the tryptophan operon (and what kind of gene expression it is)

Tryptophan operon is repressible - genes are turned off when tryptophan is available and turned on (de-repressed) when tryptophan is absent. Transcriptional level

Simultaneous vs. Sequential Regulation

Simultaneous: a single regulator (TF) regulates multiple genes at the same time. Sequential: a single regulator or cascade of regulators (TF) regulates multiple genes in a defined temporal series of events.

What region of the mRNA is needed for initiation of protein synthesis in prokaryotes?

Shine Dalgarno sequence (SD, AGGA)

Explain how a metabolite can result in a riboswitch inhibiting start of translation

Metabolite binds and stabilizes an alternative mRNA secondary structure in the 5’ UTR; this leaves the SD sequence and AUG in a base-paired region (inaccessible) —> inhibits start of translation

Explain how a metabolite can result in a riboswitch inhibiting transcription

Metabolite binds to and stabilizes an alternative mRNA secondary structure in the nascent 5’ UTR; forms a loop that interrupts RNAP —> inhibits transcription

Factors that influence mRNA stability and half-life

Secondary structure in RNA can impact half-life; protein interactions with mRNA or its secondary structures can impact stability

If an enzyme is regulated allosterically, does it affect gene expression?

No - involves a small molecule interacting (non-covalently) with an enzyme, rapid response

Which PT regulation is used to transmit signals?

Protein modification - involves covalent addition of functional groups - can be slow or rapid

Which PT regulation is used in response to stress or altered environmental conditions?

Protein modification + Transcriptional regulation - leads to changes in gene expression as well

Generally, how are metabolic pathways regulated?

Regulation by adjusting the rate of one or more regulatory enzymes that govern the overall rate of the pathway

What are examples of allosteric effectors, and what is their function?

Products (metabolic end products or intermediates) that stimulate or inhibit regulatory enzymes

If an allosteric activator binds an enzyme, what is the resulting effect?

The active site becomes the correct conformation for the substrate, which then binds the active site

In a metabolic pathway with two pathways competing from one initial compound, how is it regulated if the cell has excess of product A but minimal product B?

Product A can act as an activator of the enzyme that makes product B; product B then acts as an inhibitor of the same enzyme

When there are high levels of tryptophan in the cell, how does attenuation act on the trp operon?

Attenuation = premature termination. The ribosome would read through without stalling, allowing the terminator loop to form, displaces RNAP and stops transcription

When there are low levels of tryptophan in the cell, how does attenuation act on the trp operon?

The ribosome stalls at tandem trp codons, allowing the anti-terminator loop to form (and inhibits the terminator loop), allowing RNAP to continue

Repression vs. attenuation

Repression can quickly shut down gene expression; attenuation allows a more gradual and fine-tuned response to changing conditions

If current levels of cAMP are low, what does that say about the amount of glucose available, and the activity of lactose permease?

There is glucose available for the PTS system, and the activity of lactose permease is blocked.

Subunits of core RNAP and function

2 alpha subunits, Beta and Beta’, Omega subunit. It unwinds DNA and polymerizes RNA

Holoenzyme subunits and function

Core RNAP + sigma subunit. It recognizes and binds the promoter

Sigma subunit functions

-Recognizes and binds the promoter

-Initiates strand separation of ds DNA

-Both steps are essential for transcription

Which part of the holoenzyme contains part of the active center for RNA synthesis?

Beta and Beta’

How does RNAP know where on the DNA to start transcription?

Promoter region has a recognition site and a binding site that are recognized by Sigma

What factors affect the ability of sigma factors to bind core-RNAP?

Concentration and affinity for core-RNAP

Which sigma factor group is associated with stationary phase and starvation, and how is it regulated?

Group 2 (sigmaS/RpoS). It is regulated by proteolysis - when not needed it is degraded by proteases, facilitated by RssB

If a bacterium needs to be motile, which sigma factor would facilitate that?

Group 3: sigma28/SigD, involved in flagellum biosynthesis

What are ECF Sigma factors involved in and how are they regulated?

They are involved in stress in periplasmic space, respond to certain environmental signals (heat, protein overproduction, misfolded proteins). They autoregulate their own expression: usually co-expressed with genes that encode anti-sigma factors which keep them inactive when there is no stimulus.

What is different about the sigma54 family from the others?

No primary sequence homology to the Sigma family; will bind promoter region in absence of core-RNAP—> binds to -12 and -24 consensus sequences

Parts of Riboswitches and their functions

-Aptamer domain - senses metabolites

-Expression platform - controls gene expression or protein translation

What do Toxin-antitoxin systems code for?

-A toxin which functions to lower bacterial metabolism(in same species) —> stable

-An antitoxin that inhibits the toxin —> degradation-prone

What is an advantage of TA systems for bacteria?

Can cause rapid growth arrest if bacteria are under stress —> results in bacterial persister cells (metabolically slow-growing cells that are resistant to antibiotics and stress)