Biol 240: Regulation

Name the 4 actions that DNA is involved in

transcription, translation, DNA repair, DNA replication

Briefly explain how transcription works in bacteria:

Sigma factor binds to RNA polymerase which forms a holoenzyme. Sigma factor guides the enzyme to a promoter region and codes RNA from DNA.

Briefly explain translation in bacteria:

small ribosome subunit finds shine-delgarno sequene and puts big ribosome subunit on top where it begins coding RNA to protein

Give two examples of environmental changes that can act as a for of regulation for microbes

changes in nutrients and availability, changes in competition

three condition-specific responses that can respond to regulation

substrate-specificity, metabolism and transport, sporulation

Key cellular enzymes are:

constitutive

constitutive genes always need to be:

on

inducible genes are:

usually off but turned on when needed

when can control of gene expression take place in the central dogma?

anywhere

What are two ways proteins can be regulated:

covalent modification or allosteric hindrance

describe the covalent modification of enzymes:

molecules can covalently modify a particular protein which changes its confirmation and therefore, also its function

describe allosteric hindrance

molecule can bind to an enzymes allosteric site which changes its confirmation, which can either activate it (allosteric activator) or deactivate it (allosteric inhibitor)

Generally, why is it a good idea to inhibit unused enzyme creation?

because it wastes energy if not inhibited

What is an operon?

transcriptional unit with a series of structural genes and their transcriptional regulatory elements

which form of control (positive vs negative) inhibits transcription in an operon and which one activated it?

negative inhibits while positive activates

How does an effector molecule regulate transcription in an operon?

through allosteric hindrance

Which side of the promoter should the regulatory protein bond to if it is designed to cause positive control?

the left side of the promoter

Which side of the promoter should the regulatory protein bond to if it is designed to cause negative control? What does it inhibit?

the right side of the promoter (blocks RNA polymerase)

True or False: all operons have only ONE promoter

false, some can have more than one

what two responses can a negative-control enzyme exhibit?

repression and induction

what is involved in the repression response?

enzyme is activated because of chemical signal, and it binds to the right side of the promoter

What is involved in the induction response?

an enzyme is automatically bonded to the right side of the promoter until a chemical signal takes it away

Which response is more likely to occur in negative-control enzymes?

more enzymes exhibit the induction response

What type of enzyme creation does the repression response negative-control enzymes mainly affect?

anabolic (biosynthetic) enzymes

What type of enzyme creation does the induction response negative-control enzymes mainly affect?

catabolic enzymes (e.g. lac operon)

What is the only type of response a positive-control enzyme can exhibit?

an allosteric molecule will activate the enzyme, causing it to bind to the left side of the promoter region

What is one example of a positive-control enzyme at work?

maltose catabolism in E.coli, wherein the maltose activator enzyme only binds in the presence of maltose

How strongly do positive-control enzymes bind RNA polymerase to the promoter?

weakly

explain what happens when a positive control-enzyme binds to the left of the promoter region

the DNA is changed slightly enough to allow RNA polymerase to bind to the promoter region and begin coding. Occasionally, the positive-control enzyme will help RNA polymerase bind if it is close enough

what is the range at which the positive-control enzyme can bind to the DNA?

up to 100 base pairs away

Two types of effector molecules:

co-inducer/co-activator, co-repressor

Co-inducer/co-activator

substance that turns on enzyme production (takes negative control enzyme away)

co-repressor

substance that binds and activates a repressor (puts enzyme there)

Where does a repressor protein bind to?

the operator region

where does an activator protein bind to?

the activator binding site

What must happen in order for the Lac-operon to activate?

all the glucose in the cell must be used up, so the Lac-operon is activated to break down lactose

what is this situation called?

diauxic growth

What is the purpose of Beta-galactosidase?

cleaves lactose (breaks it down)

What is the purpose of Permease?

allows lactose into the cell (past the membrane)

What tells the cell that lactose has entered it?

molecules of allolactose created by Beta-galactosidase

What is the name of the Lac-operon repressor protein and what negative-control response does it show?

Lacl is the name of the repressor protein, it displays the induction response

What is the name of the effector molecule of the Lac-operon and what type of effector molecule is it?

allolactose is the CO-INDUCER that binds to Lacl and removes it from the operator region

What is the only real question the cell asks with respect to the Lac-operon?

is there lactose in the cell, yes or no

What is the activator protein of the Lac-operon and what is it activated by?

the Cyclic AMP Receptor Protein (CRP) is the activator protein. It is activated by Cyclic AMP (effector molecule) that is created in response to a lack of glucose within the cell

What would happen if the glucose concentration in the cell was low, the lactose concentration was high, and the cyclic AMP concentration was also high?

allolactose would be created and bind to the Lacl repressor protein, removing it from the operator site, and cyclic AMP would bind to the CRP activator protein, which woudl allow it to bind to the activator binding site. This would allow RNA polymerase to bind to the promoter region and allow for the three genes to be transcribed, and allow for lactose to be metabolized

What would happen if the glucose concentration in the cell was low, the lactose concentration was low, and the cyclic AMP concentration was high?

the cyclic AMP would bind to the CRP activator protein, which would allow it to bind to the activator binding site. However, no lactose means to allolactose. This would allow for the repressor protein to remain on the operator site, which would not allow RNA polymerase to bind to it. Therefore, the Lac-operon genes would not be transcribed and no lactose would be broken down

What would happen if the glucose concentration in the cell was high, the lactose concentration was low, and the cyclic AMP concentration was also low?

the cell would break down glucose only because it is easier to break down, and no aspect of the lac operon would be activated at all. Therefore, no genes would be transcribed and no lactose would be broken down

What would happen if the glucose concentration in the cell was high, the lactose concentration was high, and the cyclic AMP concentration was also low?

Allolactose would bind to the Lacl repressor protein, removing it from the operator site. However, no cyclic AMP is available to bind to the CRP activator protein, so RNA polymerase cannot bind to the promoter region. Therefore, no genes would be transcribed and lactose would not be broken down

what is one other way some effector molecules could inhibit transcription?

they can bind to the repressor protein and enhance its ability to bind to the operator site (can happen in tryptophan operon)

Attenuation

Interruption of transcription after initiation but before termination

What happens as a part of attentuation?

an mRNA secondary structure (hairpin loop) that is rho-independent forms and ends translation by ribosome

what allows transcription to continue in this circumstance?

"stalling out" of ribosomes in mRNA leader sequence (not enough of a particular amino acid loaded in tRNA)

Why can't attentuation occur in eukaryotes?

because transcription and translation in eukaryotes takes place at different times, while prokaryotes have both occur at the same time

difference between rho-dependent and rho-independent transcription?

rho-dependent transcription involved a rho protein which cancels the action, while rho-independent transcription involves no rho proteins, and only a hairpin loop secondary structure

What two regions of mRNA usually bind to eachother to form the hairpin loop, and what action causes them to be transcribed?

regions 3 and 4 bind to eachother, although if region 2 is skipped then regions 3 and 4 will be transcribed

in the tryptophan operon, what level of tryptophan causes a hairpin loop to form?

high levels of tryptophan causes a hairpin loop to form, while low levels allow transcription to continue

What is a quorum?

members of a group that must be present in order to conduct business

What is quorum sensing?

the regulation of gene expression in response to fluctuations in cell-population density

how are genes regulated in quorum sensing?

chemical messengers (autoinducers) are released into the environment to inhibit perticular genes in other cells of the population when there is enough present

What is an example of an animal that displays quorum sensing?

the hawaiian bobtail squid contains aliivibrio fischeri, which work together to form the lux system, which exhibits luminescent light when grown in high density

what molecule allows for bioluminescence to occur in the bobtail squid?

N-acyl-homoserine lactose (AHL)

what protein catalyzes AHL synthesis?

Luxl protein

what determines the colour of an AHL molecule?

the length of its carbon chain

What is the name of the regulatory activator protein that binds to the "lux box" (activator binding site) in the presence of AHL?

LuxR

what does this action cause?

this action causes Luciferase protein genes to be transcribed and Luxl, which creates a positive feedback loop, thus creating more AHL

4 mechanisms that can be controlled by quorum sensing?

motility, conjugation, biofilm formation, pathogenesis

how do autoinducers play a role in competition?

they can interrupt or inhibit a control pathway in other organisms in the environment

What are two-component regulatory systems?

a system in some cells that use one protein as a sensor protein, and another to control transcription

Why would a cell have this?

because it allows the cell to easily respond to the environment using signal transduction

What is the name of the sensor and response proteins?

sensor kinase and response regulator (involved in all two-component regulatory systems)

What is an example of a sensor kinase-response regulator pair?

Histamine Protein Kinase (HPK) facilitates RNA Lymerase by phosphorylating it with ATP

What is another example of a sensor kinase-response regulator pair?

VirA and VirG, where VirA activates VirG when in similar conditions to a plant (sugar and phenolic compounds)

What is benefitial about two-component processes?

different two-component regulatory systems allow for the cell to respond to different environmental stimuli

What is chemotaxis?

movement of a motile cell or organism, or part of one, in a direction corresponding to a gradient of increasing or decreasing concentration of a particular substance.

What are the results of the experiment with Mutants and normal chemotaxic bacteria?

the mutants will not move into the rod with nutrient, but the normal bacteria will

Three steps of chemotaxis:

1 - Response to signal

2 - Control of flagellar rotation (movement)

3 - Adaptation

What is the name of the protein that responds to repellants/attractants in the "response to signal" phase?

Methyl-Accepting Chemotaxis Protein (MCP)

What does the MCPs do?

they initiate signal transduction based on if they are recieving attractants or repellants. They do a tumble movement until they reach the source of the attractant

What controlls flagellar rotation?

CheP-A proteins are phosphorylated by MCPs when attractants are low. The CheP-A proteins then phosphorylate Che-Y proteins which go to the flagella and power it. This starts a tumble motion

What does adding methyl groups to MCPs do?

it decreases the sensitivity to attractants and increases sensitivity to repellants (designed to make high amounts of attractants more significant).

What happens when there is little attractants present?

the MCP will be de-methylated so that its sensitivity to attractants is increased.

What can the CheA and CheY protein systems be called?

a two-component regulatory system

Which one is the sensor kinase and which one is the response regulator?

CheA is the sensor kinase and CheY is the response regulator

What protein must CheA interact with so that the autophosphorylation of CheA is modulated?

CheW

What does modulation of the autophosphorylation of CheA allow for?

it provides for longer sustained "runs" of motion when attractants are present

A running scenario happens when:

attractants are high in concentration

A tumble scenario happens when:

repellants are high in concentration

What is CheR in charge of?

methylating the MCPs

Does methylation ever stop?

no, even if it is demethylated methylation still happens

What is CheB-P in charge of?

demethylation of MCPs

What happens when the concentration of attractants goes up?

CheA-P and CheY-P activity decrease, the cell does not tumble, CheR continues to methylate the MCP, CheB-P activity decreases, and sensitivity to attractants decrease

What happens if attractant concentration goes down?

CheA-P and CheY-P activity increase, the cell tumbles, CheR continues to methylate the MCP, CheB-P activity increases, sensitivity to attractants increases

What happens if repellant concentrations goes up?

CheA-P and CheY-P activity increase, the cell tumbles, CheR continues to methylate the MCP, CheB-P activity increases, the sensitivity to repellants decreases

What happens if repellant concentrations go down?

CheA-P and CheY-P activity decrease, the cell does not tumble, CheR continues to methylate the MCP, CheB-P activity decreases, the sensitivity to repellants increases

What are regulons?

Sets of genes that are coordinated together, responding to the same regulatory systems

What is catabolite repression?

shutdown of several systems that utilize various nutrients when glucose is present

What is the SOS response?

multigene system for wide scale DNA repair in response to serious DNA damage

Two most important regulatory proteins that are part of the SOS response:

LexA and RecA

Why is the SOS response considered a "last ditch effort"?

because it is extremely prone to making mistakes in DNA sequencing, could cause problems (better than total DNA damage)

Which gene (LexA, Rec A) is used to inhibit the SOS system and which one activates it?

LexA keeps them all off, RecA destroys LexA and activates the SOS response