Biol 240 Topic #8: Regulation

(DNA Replication ) DNA must be retained intact yet copied to make new cells.

(DNA Transcription) DNA must be turned into multiple "working copies" (mRNA) to provide for enzymes/structural protein production.

(DNA translation) (mRNA) RNA must be read and decoded to form the enzymes/structural proteins of the cell.

(DNA repair) cellular systems must have the ability to deal with DNA damage.

(Transcription in Bacteria) transcription can only proceed when Sigma Factor is bound to RNA pol core enzyme which directs it to a promoter.

(transcription in bacteria) different Sigma Factors can direct the RNA pol core enzyme to different genes as needed.

(Transcription in Bacteria) the Rho-Dependant method of transcription termination can be described as:

A rho protein follows RNA pol core enzyme and removes it from DNA when it reaches a termination sequence.

(transcription in bacteria) the Rho independant method of transcriptional termination can be described as:

An RNA hairpin loop forms which causes RNA pol to dissociate from the DNA.

(Translation in Bacteria) the small ribosomal (30S) subunit and the Shine Dalgarno (AGGAGG) sequence can help align all the machinery to correct starting locations for Translation.

(Translation in Bacteria) Multiple Shine Dalgarno sequences allow bacterial mRNA to be polycistronic.

(Regulation) One form of bacterial regulation is different environmental conditions. Such as:

1) changes in nutrient and availability

2)changes in competition.

(Regulation) Bacterial regulation allows for condition-specific responses. Three examples are:

1) substrate-specificity (glucose when available, lactose when absent)

2) metabolism and transport

3) sporulation

(Regulation) In bacteria, Key cellular enzymes are constitutive (always on) for processes such as TCA cycle or ATP synthases.

(Regulation) The making of all bacterial enzymes all the time is impractical Because an excess amount of protein production can be costly.

Bacteria have their cellular activities regulated at multiple levels.

(Regulation) Bacterial cells do not require all gene products at all times such that inducible genes are only required at particular times. However, constitutive genes are always needed to be "on".

(Regulation) The basic control of bacterial genes expression can take place on the level of transcription, translation, or post-translation.

(Protein Regulation) allosteric regulation is when a protein's activity is inhibited or activated (increased) due to the binding of an allosteric effector molecule.

(Protein Regulation) allosteric protein regulation is enabled through the binding of a non-substrate molecule at a site away from the active site.

(Protein Regulation) allosteric regulation results in the conformational change of the protein such that the substrate can no longer bind.

In allosteric protein activity regulation, the allosteric inhibitor is often the end product of a multistep pathway.

Many enzymes use allosteric regulation mechanisms.

(Protein Activity Regulation) Covalent modification which is the addition or deletion of certain functional groups may also alter enzyme conformations. (increases or decreases activity)

(Transcription Regulation) The Operon is a transcriptional unit with a series of structural genes and their transcriptional regulatory elements.

(Transcription Regulation) Energy is conserved by controlling the production of enzymes.

(Transcription Regulation) many control mechanisms work to prevent transcription of genes when they are not required.

(Transcription Regulation) Positive control is when allosteric protein acts to activate mRNA synthesis by binding to the ABS (upstream of promoter)

(regulating transcription) Negative control is when allosteric protein acts to prevent mRNA synthesis by binding to the Operator. (downstream of promoter)

(regulating transcription) E.Coli has genes under one,both, or neither (Positive control/Negative control)

(regulating transcription) Some operons can have more than one promoter each with their own control system.

The Negative Control of Transcription may involve specific repression or induction in response to condition.

Which two types of control systems are considered "Negative Control"?

- Repression (involves repressor protein)

- Induction (involves repressor protein)

What are three factors of Repression (Negative control of transcription)?

- inhibit transcription in response to a signal.

- minority of enzymes are controlled by repression.

-typically affects anabolic (biosynthetic) enzymes

What are the features of Induction? (4) (Negative control of transcription)

- Derepression of enzyme production in response to a signal. (removing repressor protein)

- Typically affects catabolic enzymes (lac operon)

- Enzymes are synthesized only when substrate is available

- No wasted energy

Describe Positive control of Transcription

Allosteric regulator proteins (activator proteins) bind specifically to activator binding site (ABS) of the promoter which helps binding RNA pol to DNA.

What is an example of Positive Transcriptional Control in E.coli

In Maltose catabolism in E.coli, the Maltose activator protein (regulator protein) only binds DNA in the presence of maltose (effector)

What are some features of Positive Transcriptional Control?

- Positively controlled promoter only weakly bind RNA pol.

- activator protein attracts polymerase to promoter which may cause DNA structural change

- Activator protein may interact directly with RNA polymerase

- Activator binding site may be close to promoter or few hundred base pairs away. (if far = bending)

Effectors (Effector Molecules) is the collective term for molecules that affect protein production in association with allosteric protein regulators.

(Effector Proteins ) A Co-inducer or Co-Activator is a substance that turns on enzyme production.

(Effectors - Effector molecules) A co-repressor is a substance that binds and activates a repressor.

Effectors interact with DNA-Binding proteins (repressor or positive control protein)

(The Lac Operon) Glucose is easier to use than Lactose such that the lac operon is not expressed until all glucose is consumed. This is an example of Diauxic growth (two phases of growth).

Lac Operon Structural Genes: (4) (Also include protein they make)

Lac Z - B-galactosidasse

Lac Y - Permease

Lac A - B-galactoside transacetylase

Lac I - LacI repressor

What are the features of the Lac Operon? (4)

-multiple control elements on both the DNA and accessory proteins

- inducible expression

-system is only turned on when needed (only used when no glucose and only lactose)

- components allow use of lactose sugar

Brefiely describe the purpose of B-galactosidase and Permease:

B-galactosidase - cleaves B-1,4 linkage of lactose (makes glucose and galactose and allolactose)

Permease - allows lactose into the cell

(Negative Control of Lac Operon)

-Repressor protein (LacI) binds to operator which blocks RNA polymerase and stops transcription

- Effector molecule (allolactose) induces transcription by inhibiting binding of the repressor to the operator (lactose must be present)

(Positive Control of Lac Operon)

Activator Protein (cyclic AMP receptor protein - CRP) binds and increases transcription when effector molecule (cAMP) is present (low glucose)

What effect does cAMP have on the cAMP receptor protein?

cAMP (effector molecule) induces conformational change in cAMP receptor protein which increases its affinty to the ABS - increasing RNA pol affinity for Lac operon promoter.

(Another Negative Control of Lac Operon)

effector molecules can also inhibit transcription by binding to the repressor protein and enhancing its ability to bind to the operator(common for anabolic operons) (Example: Tryptophan operon)

Attenuation is defined as the interruption of transcription after initiation but before termination

What are the features of Attenuation?

- Control of transcription by RNA secondary structure (mRNA)

- Interaction between translation and transcription processes

- Attenuation cannot occur in eukaryotes (translation and transcription are very spaced apart)

In Attenuation, if ribosome quickly follows RNA polymerase, rho-independent terminator hairpin RNA loops are formed in the leader sequence and the polymerase detaches (enough amino acid is present) (termination occurs at region 2 and 3)

In Attenutation, "stalling out” of ribosome in mRNA leader sequence (i.e., not enough of that amino acid loaded in tRNA) allows transcription to continue. (termination occurs at regions 3 and 4)

(Quorum sensing ) A Quorum are members of a group that must be present in order to conduct business.

What are 4 features of Quorum Sensing?

- chemical signalling system that allows microbes to communicate with each other

- Regulates gene expression based on population density

- Cells release autoinducer molecules into environment as population density increases

- Detection of changes in autoinducer levels causes regulation of gene expression.

1) regulation of gene expression based on population density. **

2) Positive feedback - if more population, more autoinducer

3) Rapid induction - turns on/off quickly

4) Links cellular behavior to population density

5) Coordinates SUPER expensive, additive processes

6) Partakes in interactions with eukaryotes

(Aliivibrio Fischeri Quorum) Lux is a prototypical quorum-sensing system found in Aliivibrio fischeri.

What are 2 features of Aliivibrio Fischeri?

- Aliivibrio Fischeri live freely or in symbiosis with the Hawaiin bobtail squid

- Aliivibrio Fischeri cell only emit light (via the enzyme luciferase) when in the light organ of the squid.

What are the features of the Lux prototypical quorum sensing system in Aliivibrio Fisheri?

- When cell grown to high density, the cells produce lots of N-acyl-homoserine lactose (AHL)

- AHL activates luminescence

- LuxI protein catalyzes AHL synthesis

What is the Positive control of transcription aspect of Aliivibrio Fischeri?

- LuxR, a regulatory transcriptional activator, interacts with AHL when it reached high enough concentration.

- LuxR binds to "lux box" DNA regulatory site (ABS)

-leads to transcription of luciferase protein genes and luxI, which creates positive feedback loops forming more AHL .

- autoinducer molecules may play a role in competition

- motility

- conjugation

- biofilm formation

- pathogenesis (all viruses use quorum sensing)

How may autoinducers of quorum sensing play a role in competition?

interrupting or inhibiting a control pathway in other organisms in the environment.

What are the two main features of Two-component regulatory systems?

1 - detecting what happens outside (past membrane)

2 - sending a signal inside to tell someone (signal)

What are the features of the Two-component Regulatory System (4)?

- Can use one protein as a sensor and another to control transcription

- Allows for cellular response based on the environment

- Signal transduction induced inside the cell alters it to respond appropriately

- Important to protect against Virulence

What are the two components of the Two-component Regulatory System?

- Sensor Kinase

- Response Regulator (RR)

1 - Membrane bound

2 - Detects environmental stimulus (events)

3 - Detects signal then phosphorylates a response regulator

(Two-component Regulatory System) What the functions of the Response Regulator?

- regulates transcription by altering DNA (positive or negative control)

- some can be either activator or repressor proteins

- phosphatases can come and remove phosphate or it can remove it by itself.

1 - vir genes found on the Ti plasmid are only expressed under conditions similar to a plant wound site

2 - virA (sensor kinase) /virG (response regulator) are required for expression of the other virulence genes (oncogenes and opine genes)

(Two-component regulatory systems) Different two-component regulatory systems can allow microbes to respond differently to environmental stimuli.

(Two-component regulatory systems) With The Pairing of particular HPKs (Histidine protein kinases) and RRs, cells can better control which genes are expressed in response to signals from the environment.

What are the features of Chemotaxis? (4)

- a complex bacterial behavior controlled by shifts in protein activity

- chemotactic bacteria sense changes in chemical gradients over time

- changes induce altered direction and duration of flagellar rotation, leading to directed movement over time.

-This is a modified two-component regulatory system.

What are the features of "Studying Chemotaxis using Mutants"?

- normal chemotaxis microbes are isolated using a capillary tube filled with nutrients such that these microbes will move into the tube.

- mutants of chemotactic proteins will remain outside the tube.

What are the three steps of Chemotaxis Regulation? (3)

Step 1: Response to signal

Step 2: Control of flagella rotation

Step 3: Adaptation

(Chemotaxis Regulation) (2 parts)

Describe Step #1: Response to signal

MCPs (Methyl accepting chemotaxis proteins)sense specific attractants/repellents and decide whether to or no initiate signal transduction.

If cell is moving in wrong direction, the concentration of attractants decrease and signal transduction occurs (tells cell something is not right).

(Chemotaxis Regulation)What are the steps of "Controlling flagella rotation)

1- MCP initiates signal transduction telling something is not good outside cell

2 - CheA -P is phosphorylated

3 - CheY is phosphorylated by CheA-P

4- CheY-P interacts with motor proteins to cause tumbles

5 - CheZ eventually takes phosphate group off and stops the tumble.

(Chemotaxis regulation) What are the features of Adaptation (step #3) ? (3)

- Feedback loops allowing system to reset itself

- Allows temporal detection of signal concentration

- requires modification of MCPs by methylation

Briefly describe the modification of MCPs during the "Adaptation" step of Chemotaxis (2 parts)

CheR keeps giving MCP methyl group to make less sensitive to attractants (when there is high number of attractants in environment)

CheB takes methyls off from MCP to make more sensitive to attractants (when bacteria has moved too far away from attractants).

When an MCP is highly methylated, it is least sensitive to attractants but highest sensitivity to repellents.

Regulons are sets of genes that are coordinated together and respond to the same transcriptional regulatory systems.

(Regulons) Catabolite repression system is the shutdown of several systems that utilize various nutrients when glucose is present.

(Regulons) SOS response is a multigene system for wide scale DNA repair in response to serious DNA damage (Ex: UV light)

(Regulon) What are the two most important proteins for the SOS response system (what are their functions as well)

LexA: DNA - binding transcriptional repressor which binds to operator and turns off SOS gene transcription when everything is ok.

RecA: De-repressor which binds to ssDNA (DNA damage) and cleaves LexA repressor. (SOS gene transcription begins)

When the SOS regulon system begins SOS gene transcription, more lexA and recA are transcribed.

(Alternating Sigma Factor) In bacteria, the use of different sigma factors directs RNA pol to certain genes.

(Alternating Sigma factors) Most E.coli promoters are recognized by sigma - 70

(Alternative Sigma Factors)

Sigma -54 : nitrogen utilization genes regulator

Sigma - 32: heat shock protein gene regulator

Sigma- 38: general stress respone gene regulator