Microbiology 2 - 2.6 Gene Expression

Why are genes regulated?

Because not all gene products are needed by the cell at all times

What is gene regulation?

The process of which the expression of genes is turned on and off at different times and under various conditions; ensures the cell won't waste its energy on making gene products (proteins) when its not required

What are the two types of gene expression?

1. Constitutive expression

2. Inducible expression

What is constitutive expression?

Constant expression of genes that encode products ALWAYS needed by the cell; housekeeping genes

What is inducible expression?

Expression of genes that encode products only when they are needed by the cell; seen when the cell is in a particular environment (like a stress response)

What are 3 ways to regulate enzymes? Which of the 3 are post-translational?

1. Allosteric regulation (post-transitional)

2. Covalent modifications (post-transitional)

3. Transcriptional regulation

What is allosteric regulation?

An effector molecule (helper molecule) binds reversibly to the enzyme's allosteric site (any site other than the active site), causing a conformational change in the enzyme

What are examples of effectors?

Activators (activate the enzyme, like fructose 1,6-biphosphate) and inhibitors (inhibit the enzyme, like ATP)

What is covalent modification?

Regulates enzymes by adding or removing a chemical group to or from the enzyme

What are the different types of covalent modification? What enzymes are responsible?

1. Phosphorylation by kinases

2. Dephosphorylation by phosphatases

3. Acetylation (addition of an acetyl group, COCH3) by acetyltransferases

4. Glycosylation by glycosyltransferase

What is transcriptional regulation?

Controls which genes are expressed at the genetic level

What is an operon?

Cluster of genes under the control of a single promoter, found mostly in prokaryotes

What is a promoter?

Region of DNA where RNA polymerase binds to begin transcription

What is an operator?

DNA sequence that transcription factors (regulatory proteins) can bind to

The ____ is not part of the operon and codes for regulatory proteins

Regulatory gene

Regulatory proteins include...

Repressors and activators

____ genes code for specific polypeptides

Structural

What are the two types of control operons?

Negative and positive

What is a negative control operon?

Regulatory mechanism involving a repressor that blocks transcription

For negative control, what are the two molecules that aid in this mechanism?

Repressors and effectors

What are repressors?

Molecules that bind to the operator on the DNA, initiating transcription; they do this by blocking RNA polymerase

What are effectors?

Molecules that bind to the activator or repressor proteins, modifying their gene regulation activity

What are two effectors used for negative control?

Inducers and corepressors

What are inducers?

Molecules that disable a repressor, thereby INCREASING transcription; turn on transcription by inhibiting repressor

What is the inducible pathway?

Involves inducers and catabolic pathways, such as the lac operon

What are corepressors?

Molecules that enhance binding of a repressor protein to an operator, thereby DECREASING transcription; turns off transcription by enhancing the repressor

What is the repressive pathway?

Involves corepressors and anabolic pathways, like the trp operon

The lac operon is a negative inducible system. List the 7 factors necessary for this pathway.

1. LacI (codes for lac repressor protein)

2. P (promoter)

3. O (operator)

4. LacZ (codes for beta-galactosidase)

5. LacY (codes for permease)

6. LacA (codes for transacetylase)

7. Inducers (effector molecule, in this case allolactose)

The negative inducible system involving the lac operon functions to?

Catabolize lactose into glucose and galactose (lactose metabolism)

Beta-galactosidase is coded by LacZ. What is this enzymes function?

Cleaves lactose into the monosaccharides, and converts lactose into allolactose

Permease is coded by LacY. What is this enzymes function?

Membrane protein that allows for lactose uptake into the cell

Transacetylase is coded by LacA. What is this enzymes function?

Transfers acetyl group to non-metabolizable sugars

The inducer functions to do what in the negative inducible pathway?

Binds to repressor and prevents the repressor from binding to the operator to induce transcription

Allolactose is the inducer for the inducible pathway. What is its function?

Signals to the cell that lactose needs to be metabolized; binds to the repressor and prevents it from binding to the operator

In the normal state when lactose is not present, the repressor is...?

Typically bound to the operator; so transcription does not occur

The trp operon is a negative repressible system. What are the 6 factors necessary for this pathway?

1. trpR (repressor protein)

2. P (promoter)

3. O (operator)

4. trpL (codes for leader peptide)

5. trpE (codes for anthranilate synthase)

6. Corepressor (effector molecule, in this case tryptophan)

The function of the negative repressible system is to...

Anabolize (synthesize) tryptophan

What is the function of the leader peptide, which is coded by trpL?

Regulates trp gene transcription by containing an attenuator sequence (which promotes the premature termination of transcription)

trpL is a structural gene, T/F?

False

What is the function of anthranilate synthase, which is coded by trpE?

Part 1 of a 2 part complex that synthesizes anthranilate (the first intermediate formed during tryptophan synthesis)

What is the function of tryptophan as an effector molecule?

- As trp levels rise in the cell, trp acts as a corepressor and binds to the repressor; this causes a conformational change, which then causes the repressor to have a higher affinity for the operator sequence (repressor binds to operator -> transcription stops)

- AKA when trp levels get too high, it causes the repressor to bind to the operator and cease transcription

What is the typical state for the repressible system?

Repressor is not bound to the operator - trp operon is transcribed

What is positive control operator?

Regulatory mechanism involving an activator that increases transcription

What are activators for positive control?

Molecules that bind to an activator binding site on the DNA, aids in the binding of RNA polymerase to the promoter to result in increased transcription

When do we see positive control?

When multiple carbon sources are available (glucose and lactose)

Which carbon source is mostly preferred?

Glucose

What is diauxic growth?

Glucose is utilized, then lactose is utilized (they are used sequentially)

The lac operon is a positive control system. What 5 factors are necessary for this pathway?

1. cAMP receptor protein (activator) - regulatory protein (CRP)

2. P (promoter)

3. O (operator)

4. LacZ (codes for beta-galactosidase)

5. cAMP (coactivator - effector molecule and signaling molecule)

What needs to occur for the lac operon to be transcribed?

Lactose must be present (repressor is NOT bound to the operator), and very low levels of glucose (high levels of cAMP)

How does a single environmental stimulus control multiple operons?

Global gene regulation; a regulatory mechanism that affects many operons or genes scattered throughout the genome

A ____ is a set of operons or genes that are affected by the same regulatory protein

Regulon

A regulon occurs due to...

Large, sudden changes in the environment such as osmotic concentration, temperature changes, and nutrient levels

What are methods for global gene regulation?

- Alternative sigma factors bind to specialized promoters of certain genes, resulting in transcription initiation

- Sigma 70

- Sigma 54

- Sigma 32

- Sigma 38

Sigma 70 is recognized by...

Most E. coli promoters (housekeeping genes)

Sigma 54 regulates...

Nitrogen utilization genes if in a nitrogen starved environment

Sigma 32 regulates...

Heat shock protein genes (proteins used to refold misfolded proteins in high temps)

Sigma 38 regulates...

General stress-response genes (if nutrient levels are too much or too little)

Regulatory RNAs are...

Small, non coding RNA molecules that often affect gene expression by interacting with existing mRNA

What are the 3 types of regulatory RNAs?

1. microRNA (miRNA)

2. small interfering RNA (siRNA)

3. antisense RNA (asRNA)

What is miRNA?

- Double stranded RNA

- Interacts with DICER and forms a complex (RNA induced silencing complex) that facilitates RNA interference (gene regulation)

What are the two ways miRNA regulates gene expression?

1. Incomplete complementary to target mRNA (binds mRNA to prevent ribosome from translating)

2. Complete complementary (miRNA facilitates cleavage of target mRNA and degradation)

What is siRNA?

- Double stranded RNA

- Interacts with DICER; forms complex (RNA induced silencing complex) that facilitates RNA interference (gene regulation)

What is the one way siRNA regulates gene expression?

Complete complementary (facilitates mRNA cleavage and degradation)

What is asRNA?

- ssRNA

- Binds to target mRNA and provides a physical block that prevents ribosome from translating

- Triggers degradation of mRNA

mRNA can also be controlled through mRNA ______

Secondary structures

An example of an mRNA secondary structure would be...

Attenuation

What is attenuation?

Transcription is prematurely stopped; this is caused by a hairpin loop formation

With low trp, transcription...

Proceeds

With high trp, transcription...

Stops